KE0026 (Bke1) labs and tutorials - Uppsala...

KE0026 (Bke1) labsand tutorials

Version 2004-03-19

© 2004 Department of Molecular Biology/SLU

KE0026 Biochemistry Labs & TutorialsThis is the starting page for the KE0026 biochemistry labs and tutorials. The labs include both wet labs and computer labs. In the wet labs you will learn how to use basic biochemistry lab equipment and techniques. In the computer labs you will learn how to use different computer tools to study biochemical problems, e.g. graphics programs to look at and analyse protein structures, and data base tools to extract information from data bases. Later, you will use these tools to study biochemical problems.

Please remember to bring a copy of the lab instructions to the wet labs since in general you will have access to these pages during these labs.not

GC lab rulesWriting lab reportsSample lab report (pdf format)How to make a simple graph with Excel (pdf)

Lab status

Lab and tutorial index1. (deadline: 2/4)Wet lab drivers licence2. (no report)Protein modelling tutorial3. (deadline: 14/4)Wet lab: Protein purification and analysis 1 & 24. (no report)Tutorial: Introduction to molecular graphics5. (summary

report at end of tutorial)Tutorial: Bioinformatics 1 - Comparing protein sequences

6. (summary report at end of tutorial)Tutorial: Bioinformatics 2 - Understanding protein structure

7. (deadline: 3/5)Wet lab: Enzyme kinetics8. (summary report at end of

tutorial ?)Tutorial: Enzyme structure and function

9. (no report)DNA modelling tutorial

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Copyright © 1997-2004. All rights reserved.Department of Molecular Biology SLU.

2004-03-19 13:56KE0026 Biochemistry Labs

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GÖL lab rulesYou are to all wet labs. We don't have lab coats for loan or for hire so if you haven't already got one, you need to buy or borrow one before the start of the course.

The first lab you will do is a simple " " that will familiarize you with the labs, security issues, and some basic biochemistry benchwork techniques.

required to wear a lab coat

Wet lab drivers licence

You must carry out this lab to be allowed to work in the course laboratories.

It is very important that you know where to find the fire-extinguisher, eye-wash, fire-blanket and first-aid board.

Check that the gas tap on your lab bench is closed before you leave at the end of the day. Be aware of the risk in using the micro gas-jet. The flame can easily extinguish and gas will leak out into the room.

How to handle dishes and waste: Don't use black markers whose colour won't disappear in the dishwasher. Please, use green or blue markers.Don't write directly on plastic beakers or cylinders use tape.The dishes must be rinsed after use and tape must be removed before putting it into the containers for dishes.Put tubes and other small objects into the smaller container on the sink and bigger items into the large container under the sink.Put pipettes in the bucket under the sink.

Hazardous waste such as petri dishes must be taped together and thrown in a special waste box where you also throw micro slides, Pasteur pipettes and broken glass.

Hazardous solutions must be collected in bottles marked with information about its contents. Needles and blood lancets are to be put into a jar placed on a bench.

Autoclaving of solutions, glassware, pipette tips and the like will be done centrally. Things you want to be autoclaved should be put on a special bench in lab 2.

Microscopes must be cleaned after use with benzine and lens or kleenex tissue. Put on the plastic covers when you leave the microscopes at the end of the day.

Balances: before weighing check that the balance is level by turning the adjustable feet until the air bubble is centered.Don't use the weighing room for anything else but weighing.Clean balance and bench after use and switch of the balance.

IN and OUT boxes for lab reports are found in room 200.

Welcome and Good Luck /Sol

Back to lab index


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2004-03-19 13:28GÖL rules and info

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How to write a lab reportDuring this course you will perform several laboratory experiments andtutorials that should be presented in lab reports. This page describesgeneral outlines for how such a report may be written. It is advisable thatyou read this page several times during the course. Hopefully this willhelp you to improve your writing skills.

Do not forget that you are writing an accurate account of what happenedfor anyone who wishes to read your report, not just the teacher who willmark it. You should be able to give your report to a friend who has neverseen the practical manual and he or she should be able to follow what youdid and what your results were, assuming they have some basicknowledge in the subject.

The report should contain your results, how you achieved them, and anevaluation of the results. You should demonstrate that you haveunderstood the experiment as well as how to document and present it. Tryto keep the text as short as possible without leaving out importantinformation or using a poor language. This is a difficult task but practicemakes perfect.

The layout is important for a good final result, hence the report should becomputer written with a word processing program. A recommended fontis e.g. Times New Roman. Figures should be constructed with the aid of acomputer program. Computer made figures are added directly in the textif it is possible to reduce them to a suitable size. The report is much easierto read if you don't have to flip back and forth between the main text andan appendix. Remember that figures must be numbered, have a legendand be referred to in the text.

Lab reports may be written in either Swedish or English. If you writeyour report in Swedish, the abstract should still be in English. Normallyyou leave one report per two persons i.e. one report per lab group even ifsome of the experiments were made in larger groups. Each of the twopersons handing in a joint report is individually and fully responsible forthe whole lab report. Reports may also be written individually. Make sureto keep your copy of the report at least until you have passed the course.

Generally, lab reports should include the following sections, as in theusual outline of a scientific paper. During the course you will performboth 'wet' laboratory experiments as well as computer tutorials. In the

latter case some minor changes may be made from the general rulesaccording to specific instructions given in connection to the tutorial.

Front pageThe front page contains title of the laboratory experiment/tutorial, yourname(s), group (A, B...), name(s) of the teacher(s), and the date you madethe experiment as well as the date you hand in the report.

AbstractThe abstract should give a brief (5-10 lines) summary of the entire reporti.e. the purpose of the lab, the methods you used, and the results youhave achieved. This is the section in a scientific article where you aresupposed to get the reader interested in the subject.

The abstract should always be written in English, even if the rest of yourreport is written in Swedish.

Common errors:• The abstract is sometimes completely left out.• Results and conclusions are missing from the abstract.

IntroductionIn the introduction you supply the reader with some basic backgroundwhich is important to allow the work to be placed into perspective. Youmay use the course textbook, the lab manual and lectures as references.The references are included in a reference list at the end of the report.Decide what the main purpose of the lab is. Does it introduce a newmethod or does it demonstrate a reaction mechanism?

Pictures may be included, e.g. a drawing of some equipment, a schematicpicture of a protein, a schematic overview of a biochemical pathway etcetc. Mathematical and chemical formula are also added to this section. Donot forget figure legends and numbering as well as references if you takepictures from the literature.

Common errors:• Some parts are left out.• Numbering of figures and/or figure legends are missing.

MethodsIn this section you describe equipment, chemicals and how you did theexperiment. If the work you are reporting is a computer tutorial youdescribe computer programs, data bases, and any particular data used(e.g. if you studied a protein structure identify it by its PDB ID code). Inprinciple it should be possible to repeat the experiment without access tothe lab manual. Describe in a concise way how the experiment wasperformed. Try to explain why things were done in a particular way, e.g.why a particular pH was chosen or why a particular chemical was added.Write this section the same day that you did the lab. Details are easilyforgotten already the day after.

Common errors:• Wrong tense. The methods section should be written in imperfect i.e.

past tense. Remember that the manual is written in imperative formi.e. it tells you how to do things. This means that you can't copy textdirectly from the manual.

ResultsThis is the most important part of the report where you should present theresults you obtained as clearly and concisely as possible, e.g. absorbancemeasurements, colour changes, or observations of bands on a gel afterelectrophoresis. Raw data and calculated values may often besummarised in tables but do not forget to also describe the results in thetext. Extensive presentations of raw data like long lists of absorbancemeasurements and calculations may be included in an appendix.References to figures and tables must be added both when they arepresent directly in the text as well in appendices. Do not forget to labelaxes in figures with both quantity and unit and that all tables must benumbered and have a clear heading.

Instructions for fitting data to a straight line:When you need to fit data to a straight line, use a least squares fittingprocedure. This is available in e.g. Excel, many calculators also have in-built least squares fitting procedures. Use common sense to detect outliersand decide what data points to include. You may draw the line by hand ifyou do it carefully, but you must always include the equation for the leastsquares line and an estimate of the lack-of-fit to the line. Don’t forget tolabel the axes of your plot.

Common errors:• Raw data sometimes missing and only the conclusions presented.• Results not described in the text, tables missing or vice versa.• Legends, headings and references left out.• Calculated values contain too many decimals.• Samples/series not identified but simply presented as “sample 1,

sample 2,…” or “series 1, series 2,…”. You need to specify what thesample or series is.

DiscussionState again what the goal of the lab was. Discuss how these goals wereaccomplished and how your results compare to previous knowledge. Didyou expect these results? Why? It is not correct to simply state that aresult is “wrong” or “right”. Instead you should make an effort to try toexplain why you got a particular value or result.

Equipment that was used may be commented, maybe it is possible todevelop or improve the method. Discuss possible errors. Are your resultsunambiguous or would it be possible to suggest additional experiments toexclude uncertainties?

Common errors:• You write e.g. “The experiment went very well and we got the results

we had expected” i.e. the results are not compared to other facts.

The last two sections of the lab report may alternatively be combined intoone section titled Results and discussion. The general outline should stillfollow the above mentioned recommendations.

Some of the labs include questions and discussion points. These aremeant to point out things you should think about. Use them as supportfor your reports, in particular in the results and discussion sections. Donot simply answer them one by one.

ReferencesHere you list the references you have given in your text. Each referenceshould list the author(s), year, title and page of the reference. You mayeither number the references as they follow in the text (1), (2) etc.Alternatively you indicate the name of the first author and publicationyear in the text e.g. (Smith et al., 1998). In the latter case the referencesare listed alphabetically.

AppendixThe appendix may contain documentation that is too extensive to beinserted inside the report, e.g. larger tables and calculations. Appendicesshould be numbered and referred to in the text.

Common errors:• No numbering or reference inside the report.

GeneralLeave your report in the lab report box marked "Till lärare" in room 200at GC. Lab reports must be handed in no later than 12:00 on thedeadline day for the lab. Corrected and commented lab reports will bereturned in the box marked "Till studenter" approximately one week afterthe deadline for handing in reports, or at the lab follow-up for the lab.Each lab report that is handed in on time and that is passed withoutmajor revision will give a bonus for the final exam of 1% of the totalexam points. Note that late reports will give no bonus and might not becorrected until after the course! No reports will be accepted after thefinal deadline (approximately two weeks after the end of the course).

Tom Taylor, Group A1

Sample lab report: Wet Lab Drivers Licence

15 march 2001

Teacher: Stefan Knight

Lab date: 19 March 2001Handed in: 20 march 2001

Abstract

The purpose of this experiment were two-fold. Firstly, the experiment served as anintroduction to the laboratory equipment and safety procedures. Secondly, we were tomeasure the extinction coefficient of copper sulphate at 600 nm byspectrophotometry. We also estimated the error made whilst pipetting the solutions,and this error was not allowed to exceed 5%. The extinction coefficient we measuredwas 0.942 M-1 cm-1, and the error we observed in pipetting was 2.0%, well within theallowed error margin.

Introduction

Some solutions absorb light at certain visible wavelengths more than others. Thisproperty gives the solution a colour which can be observed by the eye. Coppersulphate is blue in solution, since the Cu2+ ions absorb the red (longer wavelength)light which pass through. The amount of light absorbed is proportional to theconcentration of the solution, and the path length through which the light passes. Thisis shown by the Lambert-Beer law:

A = ε * c * l

where A is the absorbance, c the concentration (in molar), l the length in cm, and ε isa constant, known as the molar extinction coefficient.

In order to measure absorbance, a spectrophotometer is used. The spectrophotometercan be set to a precise light wavelength, for example 600 nm for red light, and zeroedusing a solution which does not absorb at the wavelength of interest, for examplewater. To measure the absorbance of a coloured solution, the solution is firsttransferred into a cuvette in which the path length (l) is known precisely. The cuvetteswe used were 1 cm in length. Plastic cuvettes are OK when measuring using visiblelight, but for measuring at UV wavelengths, special quartz glass cuvettes are required.

The extinction coefficient for a certain solution can be determined as follows. Using asolution of known concentration, a series of known dilutions is made and theabsorbance measured. A plot of absorbance vs. concentration (in molar) gives astraight line whose equation is given by the Lambert-Beer law. The slope of the line isthe molar extinction coefficient. The reason for taking many measurements ratherthan just one is to reduce the possible errors in dilution and measurement.

Methods

Materials

0.345 M CuSO4

Pipettes and pipette tipsPlastic cuvetteSpectrophotometer

Methods

The experiment was performed according to the instructions given in the labinstructions [1]. From the 0.345 M stock solution we made 10 dilutions by taking 0.1ml, 0.2 ml, 0.3 ml, ..., 1ml and then making the total volume up to 1 ml usingdeionised water. For volumes greater than 0.2 ml, a 1000 µl pipette was used, and forvolumes less than this a 200 µl pipette. The absorbance of each solution wasmeasured in a plastic cuvette with 1 cm path length using a spectrophotometer at awavelength of 600 nm. The spectrophotometer was first zeroed at this wavelengthusing a water blank.

Results

The results of our experiment are shown in Table 1 and the values shown plotted inFigure 1.

Table 1. The measured absorbances Abs(obs), calculated absorbances Abs(calc)based on the line of best fit, and absolute difference for the copper sulphate dilutionseries. Tube 11 corresponds to 0.345 M copper sulphate.

Tube Abs(obs) Abs(calc) |Abs(obs)-Abs(calc)|1 1.0 0.0 0.000 0.000 0.0002 0.9 0.1 0.038 0.032 0.0063 0.8 0.2 0.069 0.065 0.0044 0.7 0.3 0.103 0.098 0.0055 0.6 0.4 0.135 0.130 0.0056 0.5 0.5 0.169 0.163 0.0067 0.4 0.6 0.189 0.196 0.0078 0.3 0.7 0.227 0.228 0.0019 0.2 0.8 0.260 0.260 0.000

10 0.1 0.9 0.291 0.293 0.00211 0.0 1.0 0.327 0.326 0.001

1.808 0.037

ml H2O ml CuSO4

Figure 1. Plot of measured absorbance at 600 nm versus voulme copper sulphate

From the above data, the molar extinction coefficient for copper sulphate at 600 nmcan be calculated as follows:

Gradient of line = 0.325 cm-1 (ml CuSO4)-1

1 ml CuSO4 corresponds to 0.345 M

ε600 (CuSO4) = 0.325/0.345 = 0.942 M-1 cm-1

The residual (random error) is:

Σ(|Abs(calc)-Abs(obs)|)/Σ(Abs(obs)) = 0.037/1.808 = 0.02 (2%)

Discussion

Spectrophotometry

The goal of this experiment was to become acquainted with the laboratory and safetyaspects, and to obtain some basic experience in using the pipettes andspectrophotometers by measuring the molar extinction coefficient for copper sulphate.We obtained a value for the molar extinction coefficient at 600 nm for coppersulphate of 0.942 M-1 cm-1.

From our results, we calculated a random error of 2%. These errors are due to manyfactors, but the main reason is inconsistency in pipetting the solutions. An error lessthan 5% was acceptable for this experiment.

Other sources of errors are avoidable. The cuvette should be clean (we were carefulnot to touch the transparent sides), and always placed in the spectrophotometer in thesame direction. Bubbles in the solution should be avoided since they also scatter light.We removed small bubbles by tapping the cuvette on the lab bench. It is also not agood idea to measure absorbances which are less than 0.01 or greater than 2, sincethese values correspond to 99% and 1% transmittance respectively of the originallight beam. Most spectrophotometers have a sensitivity of this order, so the error inmeasurement is large. We measured a series of concentrations, so we proceeded fromthe lowest to the highest concentration. This minimises the error due to residualsolution left in the cuvette from the previous measurement. Ideally, the cuvette shouldbe cleaned between each measurement.

Lab Safety

Before the experiment, we had a talk about safety procedures in the lab. These areoutlined in the lab instructions [1].

References

[1] Lab 1 instructions, Bke1 Course Compendium VT 2000.

How to make a simple graph with Excel - a brief introduction

Open up Excel. A spread sheet will be displayed.

Type your values in the columns e.g.

Mr (kDa) K2,5 0,372,1 0,421,7 0,511,2 0,65

Use the mouse to mark your data

Find the Chart Wizard press it and a new window will be displayed

Choose chart type XY scatter and chart subtype scatter.Press next and you will see a graph displayed.Press next again and you will be able to choose between diffent options like Titles, Axes etc.Select Titles and type in appropriate values. You may also change other features if you wantto, I don't want grid lines so I go to that part and take away major gridlines.

Continue with next and then finish. In this state my graph looks like this

K as a function of lg Molecular Weight

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3

lg Mr (kDa)

K

Stefan Knight

How to make a simple graph with Excel - a brief introduction

You may now continue to format axes and the plot area by pointing to the different areasand pressing the right mouse button.For example I will go to format plot area and change the background colour to white. I willalso take away the outer border.By pointing at the data series you may also change the shape of them as well as adding atrend line. Now my graph looks like this.

Of course you may continue to play around with this figure and improve it in many ways,this was only a brief introduction.

K as a function of lg Molecular Weight

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.5 1 1.5 2 2.5 3

lg Mr (kDa)

K

KE0026 Biochemistry Labs

Lab index Next

Wet Lab Drivers Licence

Goals of the lab

The aims of this lab can be summarised as follows:

To become acquainted with the laboratories and safety routinesGain experience in experimental designPractise pipetting and the use of spectrophotometers

To get access to the course laboratories you will have to know your way about them, and know how to use a few instruments. There are also certain routines which facilitate the experiments and working in the lab. After succesful completion of this lab will you be allowed to use the VÖL laboratories.

This introductory wet lab consits of three parts. The first part is a general introduction to the facilities of the lab. It includes orientation and security. During the second part you will learn how to use micropipettes and the spectrophotometers. Finally, we will go through the guidelines in

which has been handed out as part of the lab compendium.

"General instructions on how to write a lab report"

Orientation

You will get to know where to find the ice-machine, refrigerator, freezer and -80 freezer, just as the cold-, microscope-, spectrophotometer-, and electrophoresis-rooms. Also where solutions and chemicals are stored and other storage rooms.

You will also be shown where to find hoods, incubators, water baths, and also learn about different water qualities. You will after this know where to put dirty, broken, radioactive or otherwise contaminated glassware.


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Safety

You must know:

where to find emergency eye-wash and showers, and how they work.how the hoods work and how to vary the flow.where to find the fire extinguisher, fire blankets and First Aid kit.how to handle dangerous chemicals & solutions.what to do in case of emergency.

Eating, drinking, smoking etc. in the lab is absolutely forbidden. You should always be wearing a proper lab coat inside the lab. Never wear your lab coat outside the lab. ( ?)

When dealing with acids and bases you should be wearing latex protective gloves. When working with very cold or very hot or fuming substances, special protection measures particularly for your hands and eyes should be taken.

Why

Spectrophotometry

Spectrophotometry is a good way of determining the concentration and sometimes the purity of certain solutions. The test solution is put in a plastic, glass, or quartz vessel called a cuvette, and placed before a source of monochromatic light. There is a detector at the other end of the cuvette which measures the amount of light transmitted through the cuvette.

Two sides of the cuvettes are transparent and these are the sides through which the light beam should pass. Never touch these sides with your fingers or any other material. ( ?)

The solution has to be up to a certain height in the cuvette to make sure the whole light-beam passes through the sample.

The length of the path of the light through the sample should always be the same (1 cm). In case multiple measurements have to be made, always make sure that the same end of the cuvette faces the light beam. There is a small arrow marked on the cuvette to indicate the direction.

Remember to mix the contents in the cuvette if more than one solution is added. This is easiest done by either covering the cuvette with its stopper or some parafilm and gently turning it upside-down a few times, or by gently pipetting the contents in and out.

If you use the same cuvette to measure different concentrations of some reagent or mix your solutions with the same tip or rod you should always proceed from low to high concentration. ( ?)

Do not allow air bubbles to get trapped inside the cuvette during measurements. ( ?)

When working with UV-light, quartz cuvettes have to be used, since glass or

Why

Why

Why



plastic cuvettes absorb UV light. Also remember that the UV-lamp has to be on a few minutes before it gives a stable light.

The concentration of a sample can be calculated from Lambert-Beer's law:

Where is the absorbance, the extinction coefficient, the concentration, and the length of the beam in the cuvette (1cm).

For best accuracy in the measurements, adjust the concentration of your samples so that 0.01 < < 2.00. ( ?)

= * * A e c l

A e cl

A Why

Pipetting

The tip has to be fastened firmly to achieve the right volume. Always change the tip between every new solution, or if it has become contaminated.

Never release the button on the top of the pipette without control from your thumb. Otherwise, some solution from the tip may be sucked into the pipette and contaminate it.

Avoid touching the tips with your fingers. To dispose a used tip use the appropiate button on the pipette. Use a beaker or a plastic can to gather disposed tips on your lab bench. At the end of the lab you can put all the tips in the waste basket.

If the solution you are pipetting is toxic/radioactive, put tips in the special box for toxic/radioactive waste.

Never try to take a bigger volume than the pipette is made for. Always keep the pipette in an upright position, when you are finished with a pipette put it back on its holder (don’t leave it lying down on the table!). This is to avoid pulling solution into the actual pipette.

Use the pipettes in their appropriate measuring region. This will improve the accuracy.

Practical: Design an assay for hemoglobin concentration

You are supplied the following solutions:

Bovine hemoglobin, 0.1 mg/ml dissolved in deionised water, centrifuged to remove solid materialTwo centrifuged samples of bovine hemoglobin at two different unknownconcentrations

Your mission is as follows:

Develop a spectrophotometric assay for the determination of hemoglobinconcentration. Using this assay, measure the concentration of molar



hemoglobin in the two samples.

Hemoglobin is a molecule which transports oxygen and carbon dioxide in the blood. The whole molecule is made up of four protein subunits which have a relative molecular weight of 16 000 each. All four subunits carry a heme group which contains an iron atom. The heme group has a characteristic spectrum with absorbance peaks at certain wavelengths. This property gives hemoglobin, and blood, its characteristic red colour.

This experiment and the lab report may be done in groups of two. Before you leave at the end of the practical, you are expected to show the results of your experiment to one of the teachers. If your results are wildly different from those we expect, you may be required to repeat the experiment. Bearing this in mind, it is very important to be accurate when pipetting solutions! There will be a group discussion before you start, during and after your experiments.

Supplemental information:

Risks and protection:

Chemicals:

Organisms:

Radioactivity:

Other:

Lab index Next

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

Lab by , , and Enrique Carredano Devapriya Choudhury Tom TaylorPage created 98.08.13 by [email protected]

Page updated 2001.02.08 by [email protected] © . All rights reserved.Department of Molecular Biology SLU.




Previous Lab index Next

Protein modelling tutorial

Litterature: Stryer chapter 3; Horton et al. chapters 3 and 4Materials: Push-fit molecular models from Nicholson, Labquip, England

The aim of this practical is to increase the understanding of three-dimensional structures of macromolecules. You will use a standardized system which consists of moulded plastic units indicating atoms or groups of atoms which can be rapidly assembled into high molecular weight molecules. Interatomic distances are automatically set by the depth of the socket in each unit. The scale is 1 cm = 1 Å (0.1 nm). An advantage with this system compared to computer graphics which you will also use during the course is that you get a much better 'hands on' feeling for atom connections, distances, sterical restraints etc.

Introduction

We will start this part of the excersise by identifying all the atoms in the units used to build the polypeptide backbone. This will help you to determine the direction of the polypeptide chain (N- or C-terminal) as well as the positions of side chains, etc.

The polypeptide backbone is made by connecting two different types of pieces. One represents the peptide bond and is constructed as a planar amide group with a socket (hole) in the carbon atom and an arm on the nitrogen for connection to the alpha carbon (C ). It is not possible to rotate about the peptide bond which is between the carbon and the nitrogen. The second piece is a tetrahedral carbon that represents the C .

As you can see from the figure below the peptide bond unit contains parts from residues - it has the carboxyl portion from one residue and the amino group from the following residue. There are two possible non-identical positions for the side chain (=R). Only one is correct for an L-amino acid. On page 43 in Stryer (53-55 in Horton) you will find a description of that.

Units for the polypeptide backbone

a

a

two


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Each group will get three segments of poly-glycine sequences. Each segment has a common motif of secondary structure: parallel -strands, antiparallel -strands and an -helix starting and ending with a loop. A few hydrogen bonds are marked with plastic tubing. We shall in detail study - and -structures and connect the segments to a protein structure with the topoplogy shown in the at the end of these instructions. In the figure, arrows represent -strands and the cylinder an -helix. Follow the instructions below and build your molecule at the same time.

Model building and questions

bb a

ab

figureb a

1. Study the -structure segments. Look carefully at the of the strands (N- and C-terminals are labeled in the figure) and the pattern of hydrogen bonds (Stryer p. 59-60; Horton p. 93).What are the differences between parallel and anti-parallel -strands?Which atoms are involved in hydrogen bonding between the -strands?

b direction

bb

2. Connect the anti-parallel strands with a -turn (Stryer p 60-61, fig. 3.42; Horton p. 93 fig. 4.18) to a -hairpin. Which amino acids are most common in -turns, and why?

bb

b

3. Study the -helix. Only a few hydrogen bonds have been indicated by plastic/rubber tubing. How many hydrogen bonds are formed from the first to the last marked bond in your helix ? What is the direction of the carbonyls -- are they pointing towards the N- or C-terminus?

a

4. What configuration of amino acids do you find in proteins (L- or D)?Mark with a few methyl groups (grey tetrahedrons) the positions of the side chains.Which amino acid has only a methyl group as a side chain and what properties does it have?

5. You are now going to connect the secondary structure elements toa peptide chain. For this exercise you need to look at the below. Remember that the N- and C-terminals have been 'labelled' with blue and red pieces, respectively.

continoustopology diagram

a. Connect the parallel -strands with the a-helix to a compact entity.bb. Assemble the resulting pieces (now only two) to form one

continous peptide chain in such a way that one of the parallel -strands is anti-parallel to one of the other antiparallel strands. Connect with a -turn and mark the H-bond in the turn with the

b

b



tubing.c. Use the topology diagram to check that your model is correct.

6. Now it is time to add some side chains.Insert the following three sequences taking into account the properties of the side chains (size, hydrophobicity, charge etc.)

gly-gly-pro ala-ser-leu lys-gln-asp

7. When a new model of a protein has been built it is common to compute a so called Ramachandran plot. Why do we do that? (Stryer p. 56 fig. 3.28; Horton p 88-89).What is a peptide plane?Which bond in the plane is "stiff", and why?Which are the only two torsion angles that can be rotated?Read the approximate allowed values of phi and psi within a

-strand, -helix and for the amino acid glycine. b a

8. Which different interactions are formed within a protein?

9. What is a -twist (Stryer p. 60 fig 3.40)? Explain and make a -twist on the -pleated sheet of your protein model.

b bb


[2][1] [3] [4] [5] [6] [7] [8] [9] [10] [11]

Lab by and Ulla Uhlin Margareta IngelmanPage updated 2002.05.08 by [email protected] © . All rights reserved.Department of Molecular Biology SLU.





Wet lab: Protein purification and analysis

The purpose of this practical is to demonstrate different types of column chromatography that are used in the purification of proteins. We will also show how to do an analysis of protein fractions with SDS-polyacrylamide gel electrophoresis.

The following experiments will be done:1. of proteins by .Determination of native molecular weight gel filtration2. of a protein solution by .Desalting gel filtration3. on an column.Packing of and separation ion exchange4. : Affinity chromatography Binding of trypsin to a Benzamidine

Sepharose column.5. on an and determination

of their subunit molecular weights.Separation of proteins SDS-polyacrylamide gel

An example of a typical purification scheme is given in the figure below. This particular sequence of steps is of course not applicable in all cases which often means that a unique purification protocol has to be developed for each new substance you wish to isolate. Most important is that the steps complement each other and that the degree of purity increases each time. The number of steps in the protocol depends on the state of the starting fraction and on how pure you want your substance.

Introduction

Crude cell extract | Streptomycin sulfate precipitation (removes nucleic acid) | Ammonium sulfate precipitation | Hydrophobic interaction chromatography (HIC) | Ion exchange chromatography | Gel filtration

To get a good recovery of the substance i.e. minimizing losses it is desirable that each step is as specific as possible. To check purity and yield you may use absorbance measurements, various types of electrophoresis and preferably also some kind of activity measurement.The column chromatography part may be performed in different ways more or less manually. In the course practical we will use both manual ways as well as a system with pump and fraction collector. For more routine


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purifications this system can be built out to monitor absorbance etc. and quite often one uses a FPLC which is a programmable system with more powerful pumps.

Gel filtration is used to separate proteins of different sizes. You may also determine the native molecular weight of a protein by this method since there is a linear correlation between the elution volume of proteins and the logarithms of their molecular weights (MW) (see ). The system contains two phases, one stationary and one mobile. The stationary phase usually consists of a cross-linked polysaccharide which forms porous beads. The mobile phase normally consists of a buffer. The separation depends on the ability of molecules to enter the pores. Smaller molecules can diffuse into the beads and move more slowly down the column. Molecules are therefore eluted in order of decreasing molecular size. By varying the degree of cross-linking the gels are optimized for different molecular weight ranges.

Gel filtration

experiment 1

The result from a gel filtration experiment is often plotted as the variation of substances eluted as a function of the elution volume, Ve (see figure below). Ve is however not the only parameter needed to describe the behaviour of a substance since this also is determined by the total volume of the column and from how it was packed.By analogy with other types of partition chromatography the elution of a solute may be characterized by a distribution coefficient (Kd). Kd is calculated for a given molecular type and represents the fraction of the stationary phase that is available for the substance. In practice Kd is difficult to determine and it is usually replaced by Kav since there is a constant relationship between Kav:Kd. Kav is obtained from

Kav = (Ve-V0)/(Vt-V0)

The total volume of the column (Vt) is simply calculated from x r x h and the void volume (V0) is determined by passing a large substance that does not interact with the beads (like blue dextran) through the column.

Elution profiles

p 2

The ability to reversibly bind molecules to immobilised charged groups is used in ion exchange chromatography (IEC). Which type of charged group one choses - positive or negative - depends on the net charge of the protein which in its turn depends on the pH. IEC is maybe the most commonly used technique today for the separation of macromolecules and is almost always

Ion exchange chromatography



included as one of the steps in the purification protocol. The experiment may be divided into four different parts:

1. Equilibration of the ion exhanger in a buffer in such a way that the molecule(s) of interest will bind in a desirable way.

2. Application of the sample. Solute molecules carrying the appropriate charge are bound reversibly to the gel. Unbound substances are washed out with the starting buffer.

3. Elution with a gradient of e.g. NaCl. This gradually increases the ionic strength and the molecules are eluted. The solute molecules are released from the column in the order of the strengths of their binding i.e. the weakly bound molecules elute first.

4. Substances that are very tightly bound are washed out with a concentrated salt solution and the column is regenerated to the starting conditions.

This is a type of adsorbtion chromatography in which the component to be purified is specifically and reversibly bound to a ligand that has been immobilized on a matrix. Any component may be use as ligand as long as it can be covalently attached to the chromatographic bed material. Examples of this type of chromatography is antigen-antibody, enzyme-substrate analogue etc.

Affinity chromatography

Sodium Dodecyl Sulfate-PolyacrylAmide Gel Electrophoresis (SDS-PAGE) is an excellent and commonly used method to analyze purity and homogeneity of protein fractions. It may furthermore be used to estimate the molecular weight of protein subunits.In general, fractionation by gel electrophoresis is based on differences in size, shape and net charge of macromolecules. Systems where you separate proteins under native conditions cannot distinguish between these effects and therefore proteins of different sizes may have the same mobility in native gels. In SDS-PAGE this problem is overcome by the introduction of an anionic detergent SDS which binds strongly to most proteins. When hot SDS is added to a protein all non-covalent bonds are disrupted and the proteins aquire a negative net charge. A concurrent treatment with a disulfide reducing agent such as -mercapto ethanol or DTT (dithiothreitol) further breaks down the macromolecules into their subunits. The electrophoretic mobility of the molecules is now considered to be a function of their sizes i.e. the migration of the SDS-treated proteins towards the anode is inversely proportional to the logarithms of their molecular weights, or more simply expressed: Small proteins migrate faster through the gel. Compare this with the situation in gel filtration.The polyacrylamide gel is formed by co-polymerization of acrylamide monomer CH =CH-CO-NH and a cross-linking monomer N,N'- methylene bisacrylamide, CH =CH-CO-NH-CH -NH-CO-CH=CH (bisacrylamide). To polymerize the gel a system consisting of ammonium persulfate (initiator) and tetramethylene ethylene diamin (TEMED) is added to generate a free radical. TEMED causes free radical generation from the ammonium persulfate which in its turn catalyzes the polymerization. The concentration of the monomers may be varied to give gels of different density. Usually gels with 10-15% acrylamide are used and the ratio bisacrylamide:acrylamide is 2.7-3.3%. In addition a so called stacking gel is cast on top of the separation gel. This has a lower concentration of

SDS-Polyacrylamide gel electrophoresis

b

2 22 2 2



acrylamide and lower pH in comparison with the main gel.

A column packed with a Sephacryl gel will be used to separate macromolecules of different sizes. The native molecular weights of the proteins will be determined.

Determination of column parameters by gelfiltrationExperiment 1.

Materials:

Column (2.6 x 25 cm) filled with Sephacryl S-300. Total volume ~150 mlSeparation equipment - peristaltic pump and fraction collector with 35-40 test tubes500 ml of de-gassed 25 mM TrisHCl pH 8.0, 0.2 M NaClSpectrophotometer (UV-lamp on)3 % Hydrogen peroxide, H O2 2Sample solution that contains the following molecules:

Cytochrome c (horse heart) 2 mg/ml MW=12.000, strongly red/brownOvalbumin (hen egg white) 4 mg/ml MW=43.000, colourlessCatalase (bovine liver) 5 mg/ml MW=240.000, weakly brownishRubisco (spinach) 1 mg/ml MW=550.000, colourlessBlue dextran 0.5 mg/ml MW=2.000.000, strongly blue

Procedure:1. Equilibrate the column with 2-3 column volumes of degassed 25 mM Tris

HCl pH 8.0 containing 0.2 M NaCl. Recommended flow rate is approximately 150 ml/h. Check this by putting a cylinder at the outlet of the column and measure the volume over 5 min. Switch on the fraction collector and fill it with 30-35 test tubes. From now on pump and collector are controlled by the run/end button on the fraction collector. Use mode for collection. Plug in the value of the time that is needed to collect 5-ml fractions. This usually corresponds to about 2 min depending on the exact flow rate. Check that the fraction collector works properly (changes fraction) by letting a few ml:s of buffer go though. Stop the pump again.

both

time

2. Apply the sample by putting the end of the tubing connected to the top of the column into the test tube with your protein sample and subsequently starting the pump. The total sample volume should not exceed 1-2% of the column volume. Stop the pump just before the sample is finished. Be careful not to get air bubbles in the system.

3. Switch back to the starting buffer and start the pump again. Elute the proteins at the flow rate mentioned above. Start the fraction collector which should be filled with test tubes and programmed in a suitable manner. Use time mode for collection. A recommended fraction volume is 5 ml which corresponds to 2-2.5 min depending on the exact flow rate. Observe the separation of the different molecules and how they move down the column. Measure the absorbance at 280 nm for each fraction. Some of the proteins are coloured which makes the evaluation more simple. The catalase may be detected with H O which is cleaved to its products water and oxygen by this enzyme producing air bubbles. Note the elution volume Ve for each molecule. The elution volume for blue dextran serves as V0. Continue the washing until all proteins have eluted.

2 2



Data, results and discussion points that should be included in your lab report

1. Construct two graphs. One where you plot A280 as a function of fraction number. From this graph it should be possible to estimate Ve for each molecule. The second graph should be a plot of Kav against log MW of the proteins.

2. In which order are the molecules eluted? Why?3. How can you use the second graph to determine the molecular weight

of an unknown sample?4. Why is the absorbance monitored at 280 nm. Could you use some other

wavelength?5. Which alternative methods can you think of to detect protein in the

fractions?

This type of column can be purchased ready to use. It is packed with a Sephadex G25 gel (gel filtration substance) and the total volume of the column is 9 ml. A coloured salt potassium dichromate is used to better demonstrate the desalting process.

Desalting of catalase with a PD10 columnExperiment 2.

Materials:

PD10 column with Sephadex G2550 ml 25 mM TrisHCl pH 8.0Pipettes, tips and test tubesCatalase (10 mg/ml) and potassium dichromate (1 mg/ml)3% Hydrogen peroxide H O2 2

Procedure:1. Mount a PD10 column vertically in a stand. Take off the lid and remove

the liquid on top of the filter. Also remove the seal at the bottom of the column. Equilibrate the column with 20-25 ml of buffer.

2. Put 10 small test tubes in a rack and put the column above the first tube. Apply 1.0 ml of a solution containing catalase (10 mg/ml) and potassium dichromate (2 mg/ml).

3. Elute stepwise with 1.0 ml of the buffer and collect at the same time 1 ml fractions. Note at which volumes the catalase and salt elute. The catalase is weakly coloured but may also be detected with H O like you did in experiment 1.

2 2

Results and discussion points that should be included in your lab report1. In which order are the molecules eluted?2. When do you think it is useful to make a desalting of the sample during

a purification procedure?3. Suggest an alternative method to remove the salt.

In this experiment a small ion exchange column is packed and subsequently used for the separation of catalase and cytochrome c.

Separation of two proteins by ion exchange chromatographyExperiment 3.

Materials:



Pasteur pipette and a small piece of glass wool.8-10 small test tubes1-2 ml of DEAE-SepharoseBuffer A: 15-20 ml 25 mM Tris HCl pH 8.0Buffer B: 5 ml 25 mM Tris HCl pH 8.0 containing 0.2 M NaCl.Catalase (1 mg/ml) and cytochrome c (1 mg/ml)

Procedure:1. Put a small piece of glass wool in the Pasteur pipette and mount it in a

stand. Put a beaker under the column outlet. The glass wool will stop the gel from being rinsed out of the column.

2. Add carefully approximately 1 ml of DEAE-Sepharose to the pipette. You avoid air bubbles if you apply the solution along the wall of the pipette. Equilibrate the column with 3 column volumes of 25 mM Tris HCl pH 8.0.

3. Place the column over a rack like in exp. 2 and apply 0.5 ml of a solution containing catalase (1 mg/ml) and cytochrome c (1 mg/ml).

4. Elute stepwise 1.0 ml each time with 25 mM Tris HCl pH 8.0 until the eluate is colourless. Then elute with approx. 3 x 1 ml of 25 mM Tris HCl pH 8.0 containing 0.2 M NaCl until the second protein elutes. Cytochrome c is clearly coloured and catalase may be detected as in earlier experiments.

Results and discussion points that should be included in your lab report1. Which protein binds stronger?2. What can be said about the isoelectric point for each protein?3. Imagine that you use an cation exchanger at the same pH. How would

that influence the elution of the proteins?4. Could these proteins have been separated by some other method?

Trypsin is an enzyme belonging to the serine proteinase family. Several trypsin inhibitors have been characterised and one of them is benzamidine (Figure 1). In the following experiment benzamidine has been covalently linked to Sepharose beads and this column material has been packed into small pre-packed columns. If you apply a solution containing trypsin to this column material the protein should reversibly bind to the ligand benzamidine and later be recovered by either a pH change or addition of excess free ligand to elute the protein. In our case we will decrease the pH to elute the protein.

Affinity chromatography - Binding of trypsin to the inhibitor benzamidineExperiment 4.

Figure 1. Partial structure of Benzamidine Sepharose.



To detect trypsin we will use an artificial substrate p-nitrophenyl-p'-guanidinobenzoate (NPGB). When this substrate is cleaved a strongly yellow coloured compound (p-nitrophenol) is formed (Figure 2).

Figure 2. Cleavage of the substrate p-nitrophenyl-p'-guanidinobenzoate (NPGB) by trypsin.

Materials:

Column: HiTrap Benzamidine Sepharose 4 Fast Flow (1 ml) with Luer lock adapter 1 ml syringe 12 small test tubes Buffer A = Binding buffer: 15-20 ml 50 mM Tris-HCl pH 8.0 containing 0.5 M NaClBuffer B = Elution buffer: 10 ml 50 mM Glycine-HCl pH 3.0. Sample: 0.5 ml trypsin (10 mg/ml) 1 ml NPGB (1.0 mg/ml, freshly made!)1 ml 1 M TrisHCl pH 8.0Small beaker with distilled waterWaste beaker

Procedure:

To avoid contact with buffers and other solutions it is advisable that you wear gloves during this experiment

1. Put 12 test tubes in a rack and number them 1-12.Add 100 l 1 M Tris-HCl pH 8.0 to numbers 7-11 You will later elute the protein by decreasing the pH to 3.0 into these tubes and the extra pH-8-buffer will help keeping the pH at a more physiological level.Add 1ml of buffer A to number 12. This will be used as a control tube.

m

2. The HiTrap system consists of convenient pre-packed columns that you may run either connected to a pump (e.g. FPLC) or manually with the aid of a syringe. The column is stored in ethanol and you should start by washing it with distilled water. Replace the top lid of the column with a Luer lock connection and remove the bottom nut. Fill a syringe with distilled water, connect it to the top of the column and flush it SLOWLY at a speed of 1 ml/min. A recommended wash volume is 3 ml. All the subsequent solutions are applied like this with the syringe.

3. Continue to wash the column with 5 ml of buffer A (binding buffer). Now the column is ready to use.

4. Apply 0.5 ml trypsin to the column (use the syringe speed of ~1 ml/min). Start collecting the eluted fluid in the first tube. Then leave the



column with the added protein for 5-10 min to allow the trypsin to bind to its ligand.

5. Apply 5 x 1 ml of buffer A (binding buffer). Collect in tubes 2-6.

6. Apply 5 x 1ml of buffer B (elution buffer). Collect in tubes 7-11.

7. Add 50 l NPGB to all tubes 1-12. Mix and observe any colour change. Leave the tubes for 5 minutes and observe them again.

m

8. Wash the column with 5 ml of water and put back the lids.

Presentation of results and questions:1. Does trypsin have an affinity for benzamidine? 2. What happens when you add the low pH buffer?3. Could you have used the same type of column for any other proteins?

We will separate proteins on a SDS-polyacrylamide gel and use the result to construct a relation between the migration properties on the gel and the molecular weight of the protein subunits. The gels will be precast and polymerized before you start.

SDS-polyacrylamide gel electrophoresisExperiment 5.

Materials:

Precast polyacrylamide gel plates, 8-16% in acrylamideElectrophoresis running buffer (25 mM Tris HCl, 0.2 M glycine pH 8.8, 0.1% SDS)Samples - one per person, micropipettes, tips, glovesSample disruption solution (200 mM Tris HCl pH 6.8, 30% glycerol,0.02% bromphenol blue, 2% SDS, 5 % -mercapto ethanol)bHeating block at 90-100° CElectrophoresis equipment - buffer chamber and power supplyStain solution (5% methanol, 10% acetic acid, 0.1% Comassie Brilliant Blue R250)Destain solution (same as the stain solution but without the dye), plastic vessels

Procedure:1. Mount the precast gel plates in the buffer chamber. You may run 2 gels/

chamber which makes it suitable to use one gel per large group of 4-5 persons. Fill the sample wells and buffer chambers with running buffer.

2. Prepare your samples by adding 25 l of sample disruption solution to 25 l of sample. Incubate at 90° C for 5 min. Load the samples on the gel according to instructions. The samples will 'fall' nicely into their wells due to the presence of glycerol which increases the density. Put on the top lid of the chamber.

mm

3. Connect the chamber to a power supply. The gels should be run at 20 mA/gel. A dye present in the sample solution makes it possible to observe the front moving towards the anode. Normal running time for the gel should be around 60 min.

4. Switch off the power supply and dismount the gels. WEAR GLOVES AND LAB COAT. The stain solution will stain your clothes a nice shade of deep blue if you spill it. Be careful not to break the gels (or the glass



plates). Transfer the gel to a stain solution for 30 min. Then destain in destain solution for at least h. After staining with Coomassie and subsequent destaining, protein bands appear strongly blue.

Data, results and discussion points that should be included in your lab report

1. What conclusions can be drawn about size of the proteins and the way they migrate in the gel?

2. Use the result from a well with a standard mixture of proteins to construct a graph where you plot the migration of the proteins (distance from starting point at the cathode in mm) as a function of the logarithms of their subunit molecular weights. Use the standard curve to determine the denatured molecular weights of your other proteins.

3. Why is SDS present in the gel, samples and buffer?4. The sample disruption buffer contains mercapto ethanol. What is the

function of the reductant? Would the pattern of protein bands look different if the reducing agent was omitted from the sample?

b-

5. You have used both gel filtration and SDS-PAGE to determine the molecular weights for some typical proteins. Compare the results and try to deduce whether these proteins are monomers, multimers (e.g. homo or hetero dimers?) in their native states.

This wet lab/tutorial consists of five separate experiments, all dealing with the separation (purification) and analysis of proteins. In your lab report, each experiment should be presented separately according to the general . Finish the report with a "Conclusions" section where you discuss advantages and disadvantages of the different methods.

Writing lab reports

guidelines for writing lab reports

Here is a short english-swedish dictionary of common protein purification terms

Some useful expressions



absorbance absorbansacrylamide akrylamidbeads pärlor, kulorblue dextran blått dextranbuffer buffertcatalase katalascolumn kolonn (pelare)cylinder mätglascytochrome c cytokrom cdegas avluftadesalt avsalta (ej samma sak som salting out = utsalta)dye färgämneelectrophoresis elektroforeselution elueringequilibrate jämviktagel filtration gelfiltreringgraph diagramfraction collector fraktionssamlareion exchanger jonbytarepartition chromatography fördelningskromatografipolyacrylamide polyakrylamidpotassium dichromate kaliumdikromat (K Cr O )2 2 7power supply spänningsaggregatprecast gel förgjuten gelsample provstain/destain solution färgings/avfärgningslösningtest tube provrörtest tube rack provrörsställtips pipettspetsartubing slang


Chemicals:

Organisms:

Radioactivity:

Other:


[3][1] [2] [4] [5] [6] [7] [8] [9] [10] [11]

Lab by Margareta IngelmanPage updated 2003.02.12 by [email protected]

Copyright © 1997, . All rights reserved.Department of Molecular Biology SLU.





Computer lab: Introduction to molecular graphics

The purpose of this lab is to learn how to use a graphics program called Swiss PDB Viewer, SPV for short. This is a program for visualising protein molecules, and will be used in more detail in later labs. The more you learn about the program during this lab, the easier it will be in the future, so take the time to play around a bit, and explore the different possibilities of the program.

The contains everything you need to know about the program, so whenever you have problems, this is the place where you find the answer.

In order to see something you need to give the coordinates for a protein to the program. Coordinates are usually written in files called pdb-files. The pdb-files of most of the structures solved so far are collected in alarge called the PDB. In the Structure gallery to this course some proteins and their pdb-files can be found. The structure gallery is under "self study resources" in the blue menu on the left.

Download two different structures from the structure gallery. You can choose any two you'd like to see.To do this, you go to the page for each structure, and with the pointer in the Chime window, hold down the mouse button and select "File" | "Save Molecule As.." from the popup menu.

Now find the program and start it. A window with different pulldown menues should now appear. This is called the graphics window.

To see a protein you must open up its pdb-file in SPV.Go to and read the first part on this page on

how to open a pdb-file, the part called: loading one molecule. Then open one of the two files you downloaded earlier. Click OK on any dialogs that might appear. You can close the inputlog window that opens.

One trick you should learn now is .

Press on your keyboard to center the molecule.

See anything yet? You should be able to see your protein, centered in the window. Now if you hold down the button on the mouse and move it your molecule will rotate. Read to see how to rotate, translate or zoom the proteins.If you have a three button mouse, try to press each of the buttons and move the mouse.

Do you see which button that causes which movement? Move around the

SPV manual

Getting started:

data-base

->

->

Opening a pdb-file in SPV:

-> this side of the SPV manual

how to center your molecule in the window-> =

How to rotate your molecule:

this side of the SPV manual

->


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two molecules a bit to get a feel for how to view them in all different angles, how to translate, and how to zoom in.

This is a very important window. When you can master this, half is won!!! If it didn't appear when you opened your pdb-file, you find it under the Wind-menu.Open up the control panel, and then read .

Now look at the control panel. You have a lot of columns present. Most important, you have a list of all the amino acid residues present in your protein.

Try clicking on one of the amino acids and then press return.Now select amino acids 1-10. Try also to hold down "ctrl" on your keyboard and then click on a number of amino acids that are not next to each other. See that you can add amino acids to view in this way? Center the amino acids you are looking at!

In some cases there is a letter to the left of your amino acids, either an s or an h. Click on one of them and press return. What did you see? Note that even if you only click on one h or s, all the amino acids close to this one with the same letter in front gets selected.What do you think the letters stand for?

What does the colums "Show" and "Side" mean?

Tips:To select all the amino acids in a protein, go to the select menu and choose all. To mark all the selected amino acids in the "Show" or "Side" column, click on the word "Show" or on the word "Side". To unmark all the amino acids selected for the same two columns, press "ctrl" and "shift" at the same time and click at the words.

Do you have a capital letter in the far left column? Like A, B or C? That means that you have more than one chain in your protein. If you click on one of these letters, all the amino acids belonging to that chain get selected.

At the top of the panel you have a box called "visible" and a box called "can move". Try checking/unchecking them to figure out what they mean.

Now open up the second pdb-file you saved, and center both of your molecules.How do you select wich protein to look at in your control panel? What happens if you check "can move" for only one of them?

Check out in the manual to read what you can do with the select menu.Together with the control panel you can choose exactly what you want to look at.

Use the select menu to look at: all the alpha helices your protein contains.Then look at your beta sheets.Look at all the polar amino-acids you have.

Control Panel:

this side of the SPV manual

Two important things: when an amino acid is couloured red it is selected, when it is black it isn't. If you press return on your keyboard, only the selected amino acids will be shown!!->

->

->

->

Select menu:this page

->



Finally check out how many phenyl-alanines there are.Don't forget to press "return" after each selection.

From this menu you can choose what to look at in the selected amino acids.

Choose to look at only the Ca atoms of your amino acids.Choose the slab alternative. What is it good for? Rotate your molecule and

see what happens.

This is the menu for colouring your molecule, or more often parts of your molecule. It is very well discribed in . Read that!

Play around with the colours, and try the different alternatives in the menu. When can it be useful to use them? Try colouring different parts of the protein in different coulours. A very useful option is to colour after secondary structure. Do that!Worth noticing is the option "other colour...". Here you can choose the colour you want by mixing red, green and blue (RGB). Play around a bit with this option. Also by clicking in the box to the far right in the control panel, you can select to colour a specific amino acid, or a group of amino acids.Do you notice that your colour selection only applies to the protein you have selected to view in the control panel? In this way you can colour your two proteins in different ways, so that you more easily recognise them.Try to do that!

In this menu you can choose what windows you want to have open. Look at the different alternatives. If there are any you don't understand, consult the manual.

This is the window that first appeared when you started up the program. You can see many buttons in this window with pictures explaining what they are for.Go through all the buttons and try to figure out what they mean. You should be able to do this just by clicking at the button and follow the instructions in the window. Don't be afraid to try! It doesn't matter if you screw up the whole structure, you're going to throw it away anyway at the end of the lab!! To "unclick" a button, just hit "esc" on your keyboard.

Now you have (hopefully) gone through some of the features of this program. Now just do this last exercise and show it to the lab-teacher.

Colour one of your two molecules after secondary structure. Look only at the ca-atoms and nothing else.

For the other molecule: Look only at the secondary structure, that is, those amino acids with either an h or an s in front of them. Try to colour all of the different strands and helices in different colour.Now move only one of your molecules so that it is as close as possible to the other one without touching it.

Did you make it? Great, now call the teacher and show what you just did!

Display menu:

->->

Colour menu:

this section of the SPV manual

->

Windows menu:

The graphics window:

->

->




[4][1] [2] [3] [5] [6] [7] [8] [9] [10] [11]

Lab by Jenny BerglundPage updated 2000.03.20 by [email protected] © . All rights reserved.Department of Molecular Biology SLU.





Computer lab: Bioinformatics 1 - Comparing protein sequencesIntroduction

This is the first in a series of two Bioinformatics computer labs where you will learn about how you can use databases to analyse families of proteins to learn something about how these proteins work. In this first part of the lab, you will learn how to find and align sequences, and when to be confident in these alignments, and when to be suspicious.

You will want to keep a word processor file as a "worksheet", and use it to copy and paste important information and results in as you go along. This file will make sure you have all of the necessary information needed to keep track of what you are doing. Save the word processor files frequently, in case something goes wrong. .

In the instructions for this tutorial, you will be asked to click a number of links that will take you various databases and sequence alignment tools on the web. When you click these links, they will open in a second (new) window. Use the first window to keep these instructions, and the second one to do all of your "work". That way you won't get mixed up when you try to use Netscape to "Go" or "Back"; don't close the second one, or you lose the links to previous results!

Proteins carry out many different tasks in living cells. The function of a protein depends on the exact arrangement of atoms in the folded structure, i.e. the 3D structure of the protein. The 3D structure in turn, depends on the amino acid sequence of the protein, which is coded by the gene for that protein. So we say that sequence determines structure determines function.

We can imagine that not all parts of a protein are equally important for function. For example, some amino acid residues might play a critical role in stabilising the three-dimensional structure of the protein whereas others can be changed without affecting the fold or the stability. Such amino acids are said to have a structural role or function. An enzyme needs to have an active site pocket where it can specifically bind its substrate and carry out catalytic chemistry. Amino acids involved in catalysis or binding are said to have a chemical function. Many substrates are small organic molecules (small compared to the protein), and the active site is usually a relatively small pocket on the surface of the protein. We therefor expect relatively few amino acids to have a chemical function. Since there is no need to preserve them, we expect amino acid residues that don't have a structural or chemical function to be changed during evolution. If we compare many different but related proteins we therefor expect a great deal of variation of these non-critical residues. In contrast, residues with either a structural or a chemical function are expected to be conserved, since changing these might have a very large impact on the function of the protein, most likely distroying it

Protein structure and function


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completely.

All living organisms use very similar systems to break down nutrients, extract energy, and build new materials needed by the cells. In other words, they have a lot of biochemistry in common. This can be explained if all living organisms have evolved from a common ancestral cell. Many of the enzymes and other proteins that carry out fundamental biochemical processes are found in most living organisms, and these proteins are related to each other: they are descendants of the equivalent proteins and enzymes in that first cell, and the amino acid sequences are recognisably similar. Due to this fact, proteins may be organised into families that are evolutionary related to each other.

During evolution, random mutagenesis events take place, that change the genes coding for the proteins. There are several different types of mutation that can occur. Gene duplication results in two copies of the same gene. After gene duplication, one of the genes might evolve to code for a protein with a new function as long as the other "backup" copy of the gene is still there to provide the original function. This is one of the ways that new traits and eventually species may come about during evolution. So called "point mutations" are either silent (no change in the protein sequence) or change amino acid residue. Insertions and deletions insert one or more amino acid residues into a protein, or delete one or more residues, respectively. Genes may also be "shuffled" to combine protein domains in novel ways.

Some of the changes will not have had a great effect on the well-being of the organism because they had no or very little effect on the function of the mutated proteins, so these changes are carried on into subsequent generations.

Some changes will make a protein non-functional, e.g. most mutations of active site residues in an enzyme, or mutations that prevent the protein from folding correctly. (What kind of mutation of a core residue would prevent proper folding?) If this happens to a protein that carries out an essential process, the cell (or organism) containing the mutation will die. Therefore, residues that are essential for a protein's function, or that are needed for the protein to fold correctly, are conserved over time, i.e. these residues will be identical in every protein of this type, regardless of which organism it comes from.

In summary then, if we compare the amino acid sequences of proteins that belong to the same protein family, we can learn a lot about which residues are essential for function by looking for those residues that remain the same in all of the members in the family.

Evolution

one

Oxidative phosphorylation (Stryer chapter 18; Horton chapter 14) is a fundamental process for energy production used by all cells. The process depends on transportation of electrons across a membrane which in turn generates a proton gradient which is used to drive the synthesis of ATP (you will learn more about oxidative phosphorylation later in the course). The electrons transported during oxidative phosphorylation are carried across the membrane through a series of (mostly membrane bound) redox proteins.

Cytochrome c



One of these redox proteins is cytochrome c (Stryer 18.3.3; Horton pp 224-226) (see the image on the right - to get a window where you can rotate the molecule). Cytochrome c is a soluble protein that contains a heme group whose central iron ion can undergo reversible one-electron reduction (Fe + e <--> Fe ). The heme group in cytochrome c is bound in such a way that the redox potential of the heme iron allows it to pick up electrons from one of the membrane bound protein complexes of the electron transport chain (Complex III, cytochrome c reductase) and deliver them to the next (Complex IV, cytochrome c oxidase).

We can learn about the way cytochrome c works by looking at all the mutagenesis experiments already done by nature during evolution. This is a very convenient way of doing structure/function studies since we can use information stored in databases.

click here Chemscape Chime

3+ - 2+

One can learn a lot about protein function by using the enormous amount of data available in databases. These databases not only store data; they also describe ("annotate") the data and provide tools for analysing these data. Below is a short list of some of the most important databases:

Finding information: Databases

GenBankGenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences.

Swiss-ProtThe SWISS-PROT Protein Sequence Database is a database of protein (amino acid) sequences. It contains high-quality annotations (such as the description of the function of a protein, its domains structure, post-translational modifications, variants, etc) and is cross-referenced to several other databases including Medline and the PDB.

Protein Data Bank (PDB)The Protein Data Bank (PDB) stores information about the three-dimensional structures of biological macromolecules, mostly proteins.

MedlineOnline literature database for the life sciences.

EntrezA convenient gateway to all of the databases listed below, as well as several others,



is the Entrez site hosted by ( ).NCBI (http://www.ncbi.nlm.nih.gov)

Practical

The objective of today’s work is to find the amino acid sequence for the protein we are interested in - cytochrome c, from one particular organism (in this case tuna fish). We will then search the protein structure database to find other proteins with sequences that are similar to that of tuna cytochrome c, and for which the thredimensional structure is known. We will make a "multiple sequence alignment" of a few of the sequences we find, to see what they all have in common, and in what ways they differ. Features that have been conserved throughout evolution are likely to be structurally or chemically important for the common function of these proteins.

1. Find the sequence of tuna cytochrome:

:How many cytochrome sequences did you find? How many were from tuna; do these sequences differ from each other, and if so, how?

a. Go to the Entrez site (http://www.ncbi.nlm.nih.gov/Entrez)b. Select "Proteins" in the search selector near the top of the page.c. Find the sequence of tuna (Albacore) cytochrome c.d. Copy it in FASTA format, and paste it into your "worksheet".

(Hint: Monaco and Courier are fixed-width fonts; use them to make your sequences easier to see.)

2. BLAST (Basic Local Alignment Search Tool) is a tool to search sequence databases for sequences that resemble another sequence. The sequence you use in the search is called the "query" sequence; sequences you find are called "hits." The BLAST method tries to find the longest segments of sequence from any protein in a database with significant homology to the "query" sequence. Do a BLAST search with the Albacore cytochrome c sequence:

What general types of proteins did you find? Where they mostly other

a. Go to http://www.ncbi.nlm.nih.gov/BLASTb. Go to PSI- and PHI-BLASTc. Enter the sequence (use your computers copy/paste commands,

and your "worksheet"). Select the PDB database for searching. BLAST!

d. A new page will appear telling you when you can expect the results to be ready (normally 5 seconds - a few minutes). When you want to look at your results, click the "Format!" button. A new window with your results will appear. Examine the results and note how many significant hits you found. (A significant hit is defined here as a hit with an E value better than the threshold, which is set at 0.002 by default).

e. The sequences you found can be used to search for more distantly related proteins by combining the sequence information from all of the sequences. Hit the "Run PSI-Blast interation 2" button to do this. Go back to the "formatting BLAST" page and click "Format!" to view the results. Did you find any new hits? Any new significant hits?



cytochromes, or did you also find other proteins with other functions? What types of organisms did the proteins come from? (Look at the taxonomy report to find out; the link is at the beginning of the results page).

Look at the BLAST alignments between your sequence (3cyt), and those for 1ccr, 1c2r, and 2cy3 (use your browsers "Find" command to find the sequences). What proteins and organisms do those 3 other sequences represent?

What are the reported probabilities that the observed similarities happened by chance (E values) for the alignments of 1ccr, 1c2r, and 2cy3 with 3cyt? What sequence identities do these probabilites correspond to? Do the aligned segments represent the whole proteins?

3. We can compare protein sequences to each other by aligning them, that is, putting them above each other in a way that lets us see their similarities and differences. In the alignment, we want to have identical residues in a given position in as many of the sequences as possible. For example, a good alignment of the two sequences

and

would be

Notice that in order to get a good alignment we had to shift the first sequence two residues to the right, and to "break" it up in two parts by inserting a gap in the middle. Also note how colouring residues that are identical in both sequences makes the similarities and differences easier to see. We can count the number of identical amino acid residues and divide this number with the total number of aligned residues to get a quantitative measure of the similarity: here there are 9 identical residues out of 17 compared, or 52.9% identities. In general, the longer the compared segments, and the higher the percentage identity, the more likely it is that the proteins have a common ancestor, and the more similar the protein's 3D structures will also be. (Note that comparing short segments as those above can be very misleading since the shorter the segment, the easier it is to find some similarity just by chance. When we want to compare proteins we therefore look for alignments that cover a large portion of the sequences we are comparing.)

Now align the four cytochrome c sequences (3cyt, 1ccr, 1c2r, and 2cy3). The sequence alignment program ClustalW can be used via Netscape:

GAFTLDLYVSGPRVQIT...

AAGAYSLNLYVNKKGPDIEAT...

-- FT D S-- RVQI ...AA YS N NKK DIEA ...GA L LYV GP TGA L LYV GP T

a. Go back to the BLAST report, and click on the blue link to the left of each sequence that you need. Select "FASTA" format in the selector and hit "Display" to see the FASTA format version of the sequence. Sequences in FASTA format start with a header line (indicated by a ">"), followed by the amino acid sequence in one-letter code.

b. Paste each sequence (including the header) one after the other into



Which sequences are most similar to each other? Which sequence has the most insertions/deletions with respect to our "template" sequence 3cyt? Which sequence has the fewest? Do you notice anything different about 2cy3? How many residues are conserved in all of these: 3cyt, 1ccr and 1c2r?

a single word processor file , and save the file with the format "text only" (give it a name without spaces and without special characters like *). The text after the ">" can be anything so you can change this to something that is easy for you to remember.

c. Go to .

http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html

d. Paste all of your FASTA sequences in the window (from your file!).e. Choose "ClustalW", then "submit." Save the alignment for your

report.f. Now redo the alignment with just the first three sequences (3cyt,

1ccr, 1c2r). Save the alignment for your report.

4. Now have a look at the cytochrome structures in the course. Do they all look alike? Which one(s) of these do you think 1c2r

would be most similar to?

Structure Gallery

5. Since the amino acid sequence of a protein determines its 3D structure, two different proteins can obviously not have exactly the same structure. But if the sequences are similar, we expect the 3D structures to also be similar. (Since the structure of a protein determines its function, we then also expect proteins with similar sequences and structures to work and behave in similar ways. The reverse is not necessarily true since there can be many ways of getting a particular "job" done.)

So how can we go about comparing protein 3D structures to see what they have in common and how they differ? We could try to view the two structures side by side, but this would make it very difficult to compare them. A much better way would be to put one structure on top of the other to get maximal overlap. This would make it easy to see where the proteins are similar and overlap, and where they are different.

A method that is commonly used is to overlap ("superimpose") only the C atoms of two structures. This makes sense since the C trace of a protein shows how the main chain is folded, so to say that two proteins have similar C -traces is the same as saying that they have similar folds. After having aligned two similar structures, we generally find that some segments of the protein chains overlap nicely, but that others don't overlap at all. To get a measure of similar two structures are we can calculate the root-mean-square distance (r.m.s.d.) between C -atoms in segments that overlap. The more atoms that match, and the smaller the r.m.s.d., the more similar the structures.

The structures of 1ccr, 1c2r, and 2cy3 were compared to the structure of 3cyt by superimposing C atoms. The segments where each pair of Catoms are within 3.8 Å of each other, and the r.m.s.d. for all pairs of Catoms whithin each such segment, are reported below:

a a

a

howa

a aa



Structure r.m.s.d. # of atoms residues equivalent to3cyt residues

1ccr 0.561 103 9 - 111 1 - 103

1c2r 1.357 93 1 - 10 1 - 1010 - 21 11 - 2232 - 61 27 - 5665 - 83 57 - 75

92 - 101 76 - 85103 - 114 90 - 101

Segments of 2cy3 could not be matched in this way.

Compare the results of the sequence alignments, and of the BLAST comparisons, with the structural information provided here. How are structural similarity and sequence similarity correlated?

Lecture:Lecture 6: Protein sequences, bioinformatics

Chothia C and Lesk A (1986). The relation between the divergence of sequence and structure in proteins. . :823-826

The BioTech site provides a short .

Here's a

General background reading

EMBO J 5

introduction to Bioinformatics

tutorial on sequence searching

If you want to have a copy of this program to run on your own computer, it (ClustalW) can be downloaded to your machine from this site.

Altschul SF, GishW, Miller W, Myers EW and Lipman DJ (1990). Basic localalignment search tool. . :403-10

Information about the programsInformation about the Clustal W program.

J. Mol. Biol 215


[5][1] [2] [3] [4] [6] [7] [8] [9] [10] [11]

Lab and Sherry Mowbray Stefan KnightPage updated 2003.03.21 by [email protected]






Computer lab: Bioinformatics 2 - Understanding protein 3D structureIntroduction

This is the second in a series of two Bioinformatics computer labs. The purpose of this lab is to learn how evolution, when looked at in the light of protein 3D structure, can help us understand how proteins work. We will compare a small number of cytochrome c molecules from very distantly related organisms, and from this try to figure out what parts of the molecule are most important, and why.

We will use the graphics program SWISS PDB VIEWER (SPV), which you learned to use in the lab.

As discussed in the introduction to the 1st Bioinformatics lab -, random mutations that change the sequence of a protein

will occur during evolution. We can imagine a number of outcomes of such a change. The mutation might have no effect on the structure or the chemical function of the protein. If this is the case, the change will be kept and transferred to the next generation. The mutation might disrupt the structure so badly that the protein can't even fold properly. In this case the organism will most likely die, and that change will not be transferred to the next generation. The mutation might change a residue that is critical for the protein's chemical function without affecting the overall 3D structure of the protein. When this happens, again the organism will most likely die, and the mutation will not be transferred to the next generation.

So, over a very long time, we expect residues that don't have a structural or chemical function to diverge, i.e. these residues might vary a lot when we look at the same protein from many different organisms. Residues that have either a structural or a chemical function, on the other hand, are expected to be retained during evolution. So if we compare the sequences of the same protein from many different organisms we should be able to identify the residues that are important for structure and/or chemical function by finding the residues that haven't changed. By looking at these conserved residues are in the three dimensional structure of a protein, we might also be able to say something about they are important.

To make an analysis like this most effective, we need to compare many more than just three sequences as you did in part 1 of this lab, because when we consider only a few sequences, many residues might be identical by chance rather than because they are truly necessary to maintain the function of the protein. Also, some errors may occur in how the sequences are aligned to each other, and such errors are easier to detect and correct when more sequences are used in the alignment. To save some time we have already fetched and aligned some more cytochrome c sequences from the Swiss-Prot database:

Introduction to molecular graphics

Comparing protein sequences

where

why



Tuna ---------GDVAKG KKTFVQKCAQCHTVE NG------GKHKVGP NLWGLFGRKTGQAEG YSYTDAN---KSKGIHorse ---------GDVEKG KKIFVQKCAQCHTVE KG------GKHKTGP NLHGLFGRKTGQAPG FTYTDAN---KNKGI Mouse --------MGDVEKG KKIFVQKCAQCHTVE KG------GKHKTGP NLHGLFGRKTGQAAG FSYTDAN---KNKGI Human --------MGDVEKG KKIFIMKCSQCHTVE KG------GKHKTGP NLHGLFGRKTGQAPG YSYTAAN---KNKGI Rice -ASFSEAPPGNPKAG EKIFKTKCAQCHTVD KG------AGHKQGP NLNGLFGRQSGTTPG YSYSTAD---KNMAV Yeast AKESTGFKPGSAKKG ATLFKTRCQQCHTIE EG------GPNKVGP NLHGIFGRHSGQVKG YSYTDAN---INKNV Rh Rubrum --------EGDAAAG EKVSK-KCLACHTFD QG------GANKVGP NLFGVFENTAAHKDN YAYSESYTEMKAKGL Rh Capsulatus---------GDAAKG EKEFN-KCKTCHSII APDGTEIVKGAKTGP NLYGVVGRTAGTYPE FKYKDSIVALGASGF

Tuna VWNNDTLMEYLENPK KYIPG--------TK MIFAGIKKKGERQDL VAYLKSATS- Horse TWKEETLMEYLENPK KYIPG--------TK MIFAGIKKKTEREDL IAYLKKATNE Mouse TWGEDTLMEYLENPK KYIPG--------TK MIFAGIKKKGERADL IAYLKKATNE Human IWGEDTLMEYLENPK KYIPG--------TK MIFVGIKKKEERADL IAYLKKATNE Rice IWEENTLYDYLLNPK KYIPG--------TK MVFPGLKKPQERADL ISYLKEATS- Yeast KWDEDSMSEYLTNPK KYIPG--------TK MAFAGLKKEKDRNDL ITYMTKAAK- Rh Rubrum TWTEANLAAYVKNPK AFVLEKSGDPKAKSK MTFK-LTKDDEIENV IAYLKTL--- Rh Capsulatus AWTEEDIATYVKDPG AFLKEKLDDKKAKTG MAFK-LAKGGE--DV AAYLASVVK-

Practical

You will have noted from your own alignment, and from the alignment above, that the different cytochrome c sequences are of different length, and that the sequences have been shifted with respect to each other in a few places (by the introduction of "gaps" in the shorter sequence) in order to maximise the number of identical positions.

One relevant question is then: How do these insertions (or deletions) affect the 3D structure of the protein? To find out, do the following:

1. Go to the PDB ( ) and fetch the PDB-files for the three related cytochromes you aligned in the first part of this lab (pdb id codes 3cyt (tuna), 1ccr (rice), 1c2r (Rh. capsulatus)):

http://www.rcsb.org/pdb/

a. Enter the PDB ID code in the "Enter a PDB ID:" field on the right hand side of the page; click the "Explore" button. This will take you to a summary page for that PDB entry.

b. Click the "Display/Download file" link on the left.c. In the table under the heading "Download the Structure File", click

the "X" in the upper left cell of the table to download the PDB file to your hard drive. Make sure you know you save the files!where

d. There is an "Explore" button on the left of the download window, so you can go directly to the summary page for the other structures from here. Simply enter the ID code for the next structure and then repeat the above procedure.

2. Load all three molecules into SPV.

3. Use the "Magic fit..." option in the Tools menu to superimpose the three structures. (You might have to first use the "Magic fit" on 3cyt and 1ccr, and then on 1ccr and 1c2r to get this to work - ask a teacher for assistance if you have problems). If you colour the C traces of the molecules in different colours it should be easy to see where the structures are different and to locate the "extra" residues in the longer cytochromes.

a

4. Are the structures similar to each other? Describe the tertiary structure in general terms. Do you think they are similar enough so that it would



be valid to use one of them as a "prototype" for the others? Where in the structure do you find the insertions? Does that make sense from a structural point of view? Why/why not?

We will now use one of the cytochromes (3cyt) from our alignment as a "prototype" cytochrome c structure. (Again, do you think this is a reasonable thing to do, judging from the superpositioning of 3cyt, 1ccr, and 1c2r you just did?) If we agree that this can be done, we can use 3cyt as a typical example of this type of cytochrome. By looking at where the conserved residues are in the structure we should in many cases be able to figure out they are so important that evolution hasn't changed them. What you need to do is to look at the conserved residues and try to understand their role in maintaining the 3D structure and/or the chemical function of the protein (so you need to understand a bit about what cytochrome c does – look at the introduction to part 1 of the lab and look it up in your textbook). Ask yourself: Why can't this residue be changed? What does it do? Why does it have to be just this residue type?

Before you start, paste the alignment above into your lab report, identify and mark the residues that are identical in all eight sequences. Also, prepare a table something like the one below before you start looking at the structure (use the tuna - 3cyt - sequence for numbering the residues). Then fill out this table as you go along.

why

Residue Nr Amino acid Core/surface Heme interactions Comments

Find the conserved residues in the 3cyt structure. Does the structure give you any ideas why these particular residues are conserved? Summarise your findings in your table. Comment on you think a residue has been conserved.

why

1. Where in the structure (side chain solvent exposed or buried, turns or loops, heme binding pocket...) are the conserved residues? Are the conserved residues concentrated in one or a few regions of the molecule or are they distributed randomly all over?

2. What residues co-ordinate the heme iron? Are these residues conserved? Is the heme binding pocket made mostly from conserved or from non-conserved residues? How would you like to characterise the heme surroundings – hydrophobic, hydrophilic, charged, mixed? How do you think this affects the redox potential of the heme iron compared to an isolated heme group in solution?

3. Are there any conserved residues far away from the heme (with far away we mean too distant to interact with the heme)? Why do you think these are conserved? Could they be important for the fold of cytochrome c? (Hint: some of the amino acids have special properties that have a large effect on the conformation of the main chain.)



4. As was mentioned in the introduction to the of this lab, cytochrome c carries electrons from cytochrome c reductase to cytochrome c oxidase during oxidative phosphorylation. Cytochrome c interacts with the reductase and the oxidase by binding to negatively charged residues on the surface of these protein complexes. Can you suggest what part(s) of cytochrome c might be involved in these interactions?

If you have time:

previous part

5. Cytochrome c uses a heme group to transport electrons. Haemoglobin and myoglobin use a heme group to transport and store oxygen, respectively. Look at the structure of myoglobin (get the co-ordinates from the PDB by searching for myoglobin – you will find many entries but just pick any) and

What similarities do you see? What differences do you see? Try to explain how the differences in heme binding give rise to different functions in myoglobin/haemoglobin and cytochrome c. (Tip: look at the iron ligands.)

compare the binding of heme in myoglobin and cytochrome c.


[6][1] [2] [3] [4] [5] [7] [8] [9] [10] [11]

Lab by and Stefan Knight Sherry MowbrayPage updated 2003.03.21 by [email protected]




Bke2 Biochemistry Labs


Wet Lab: Enzyme kinetics

Stryer, chapter 8 (Horton, Chapter 5 (2nd and 3rd edition))Litterature:

In order to study the effects of different metabolites and conditions on enzymes and to determine how their activity is relevant to the functioning of a cell, it is necessary to be able to quantitate their activity how well they bind to substrates, and how fast they can turn over. This is the subject of enzyme kinetics, the study of enzymatic activity.

Introduction

In this lab you will be looking at a reaction catalysed by. This enzyme recognises in di-, oligo- and poly-

saccharides and catalyses the hydrolysis of the glycosidic bond to beta-galactose. Many beta-galactosidases act on milk sugar, and those enzymes are also called Lactose is a disaccharide of galactose and glucose where galactose is bound by a beta-glycosidic bond to the hydroxyl on carbon 4 of glucose. Galactose and glucose are both aldo-hexoses and they differ only in the orientation of OH4 (the hydroxyl group on C4), which is axial in galactose and equatorial in glucose when the sugar is in its most stable chair conformation. The structure of lactose and its cleavage to galactose and glucose is shown in the figure below:

BETA-GALACTOSIDASE galactose

lactose,lactases.

Beta-galactosidases are used in large scale in the dairy industry, for example for making low-lactose milk for people with lactose intolerance. Actually the majority of the world's adult population is intolerant to lactose. The enzyme used in this lab is from , a mould fungus ("mögelsvamp"). It is mainly used for hydrolysis of lactose in whey ("vassle"), which makes the whey sweeter and easier to concentrate. You can read more about the use of beta-galactosidase in the dairy industry on these web pages:

Aspergillus oryzae

http://www.fst.rdg.ac.uk/courses/fs560/topic1/t1f/t1f.htmhttp://www.sbu.ac.uk/biology/enztech/lactase.html


Page 1 of 7file://localhost/Users/stefan/WWW/Courses/Bke1/Labs/enz_kinetics_lab.html

To make the measurements easy we will use an artificial substrate, (pNP-Gal or pNPG), in which a ,

para-(or 4-)nitrophenol, is linked by a heteroglycosidic bond to beta-galactose. When the substrate is hydrolysed, p-nitrophenol is formed. At alkaline pH the phenolic proton dissociates (the pKa for p-nitrophenol is around 9) giving a phenolate anion with an intense yellow colour that can be easily measured in a spectrophotometer. The p-nitrophenyl group can not ionise as long as it is covalently bound to the galactose, only after hydrolysis will the colour develop. Such substrates are called

p-nitrophenyl-beta-galactoside chromophore

chromogenic.

You will perform two experiments. In the first experiment you will determine the Michaelis constants Vmax and Km of beta-galactosidase for p-nitrophenyl-beta-galactoside. In the second you will perform the same experiment in the presence of an inhibitor, galactose, or another substrate, lactose. From the results you shall decide if the inhibition is competitive or non-competitive.

There are two methods of measuring the rate of a reaction. In the first, the rate of formation of a product (or of course disappearance of a substrate) is measured over a period of time, and from this the 'initial rate' is extrapolated, that is the rate of the reaction when the reaction was started and the concentration of substrate is known precisely.

The second method assumes that the amount of substrate is high enough such that its disappearance over a given period of time is insignificant (i.e. the rate of reaction is close to linear for the first stage of the reaction). After this fixed period of time the reaction is stopped and the concentration of formed product is measured. You will use this second method. The reaction is stopped by adding an equal volume of 0.5 M sodium carbonate to the reaction mixture. This will make the solution strongly alkaline. The high pH of 11-12 (you may check the actual pH if you want) will efficiently inactivate the enzyme and ensure that the colour is fully developed.

The way the rate of an enzyme-catalyzed reaction varies with the amount of substrate is described for a simple single-substrate reaction by the Michaelis-Menten equation:

where v is the rate of the reaction, Vmax the maximum rate of reaction, S the substrate concentration and Km a value related to how strongly the substrate is bound. By measuring the rate of reaction at different concentrations of substrate

v=Vmax x S/(Km + S)



it is possible to construct a straight line (by transforming the above equation, for example using the Lineweaver-Burke method) and directly calculate the constants Vmax and Km.

You are provided with the following solutions:Experimental procedures

0.1 M sodium acetate buffer pH 5.0 (NaAc), 10 ml5 mM p-nitrophenyl-beta-galactoside (pNP-Gal) in buffer, 2 x 2.5 ml1.5 ug/ml beta-galactosidase (Enz) in buffer, 5 mlA. oryzae0.5 M Na CO , 12 ml2 325 mM galactose (Gal) in buffer, 1.5 ml

In cuvettes mix the following amounts of the reagents: (volumes in ul)Prepare Blank sample

Blank

Na2CO3 500 Enzyme 100 pNPG 0 Buffer 400

The "Blank" is used to zero the spectrophotometer. Other things than substrate and prouct that are present in your solutions may absorb light and that must be compansated for. Save the blank and use also for experiment 2.

In cuvettes mix the following amounts of the above reagents (volumes in ul):Experiment 1

1 2 3 4 5 6 7 8 9 ControlBuffer 390 375 350 325 300 250 200 150 100 100 pNPG 10 25 50 75 100 150 200 250 300 300

Make sure that the solutions have reached room temperature before starting the reaction. The reaction is then started by adding 100 ul enzyme solution to the cuvettes 1 to 9, but not to the Control. After 10 minutes it is stopped by adding 500 ul 0.5 M Na CO . This can be done as follows:2 3

1. At t=0 minutes add enzyme to cuvette 1. Mix gently with the pipette.At t=0.5 minutes add enzyme to cuvette 2. Mix gently....At t=4.0 minutes start the reaction in cuvette 9 as before.

2. At t=10 minutes stop the reaction by adding 500 ul 0.5 M Na CO to cuvette 1. Mix.At t=10.5 minutes stop the reaction in cuvette 2....At t=14.0 minutes stop the reaction in cuvette 9.

2 3

When you have stopped the reaction in cuvette 9, add 500 ul 0.5 M Na CO to the , mix well and then add 100 ul enzyme solution. The is used to measure possible background absorbance in the substrate solution. The substrate itself may absorb light and there may be product, free p-nitrophenol, present already before the reaction has started. pNP-Gal can undergo hydrolysis

2 3'Control' 'Control'



at a slow rate even in the absence of enzyme and because of possible enzyme contamination in any of the solutions. Finally pNP-Gal is slowly hydrolysed in the strongly alkaline solution after sodium carbonate addition. By subtracting this background the corrected absorbance will only come from the amount of product formed by the added enzyme during the reaction time.

The absorbance of the solutions can now be measured in the spectrophotometer. Place the cuvettes so the light goes through the thickest part.

Before doing absorbance measurements always: i) Make sure that the contents in the cuvette are well mixed; ii) carefully wipe off the surfaces through which the light shall pass; iii) check that the surfaces are clear and there is no turbidity in the solution by looking through the cuvette immediately before you put it in the spectrophotometer.

The light path shall be 1 cm.

Mix - Wipe - Look - Measure

Set the wavelength to 400 nm on the spectrophotometer. Zero using the Now read the absorbances of the reaction mixtures and record the results.

'blank'.

Measure the absorbance of the and write down the value. If its absorbance is 0.02-0.03 or less the background can be ignored, but if larger, the background absorbance must be subtracted from the absorbance after the reaction. Note that the absorbance of the is the background only for the highest substrate concentration. The background for the other concentrations can easily be extrapolated, since absorbance is directly proportional to the concentration.

'Control'

'Control'

Rinse the cuvettes thoroughly with water and place them upside down to dry for use in Experiment 2.

Plot the vs. on a scrap piece of paper. This will give you some idea if any of the points are spurious. Any reaction which looks suspicious can then be repeated if it seems necessary. Show the plot to the lab teacher before proceeding with Experiment 2.

absorbance substrate volume

Before you start this experiment make sure that the results of Experiment 1 are acceptable.

In order to investigate the effect of the inhibitor on the rate of the reaction, repeat the above experiment (using the same cuvettes after having rinsed them thoroughly with water and dried them) but include in the reaction mixture 100 ul inhibitor solution, maintaining the same volume as before in the reaction mixture (i.e., add 100 ul less of the buffer). Make a quick plot as before to check if any reaction looks suspicious.

Experiment 2

For each experiment do the following:Treatment of the data

1. Calculate for each tube the concentration of pNP-Gal as it was in the cuvette at the start of the reaction (reaction volume 0.5 ml).

2. Calculate the background in each cuvette. The absorbance of the control is the background for cuvette 9. For cuvettes 1 to 8 it has to be extrapolated from the Control. See .Appendix

3. Calculate for each tube the rate of the reaction in terms of how many mol/3



dm of pNP-Gal that were hydrolysed per minute. The molar absorption coefficient of p-nitrophenol at high pH is 18,300 M *cm at 400 nm (i.e. a 1 M solution of p-nitrophenol would have an absorbance of 18,300 - if that were possible to measure). Bear in mind the following points:- The final volume is 1.0 ml whereas the reaction volume is 0.5 ml- The reaction period was 10 minutes

-1 -1

4. The table in the gives you a hint on how to treat the data.Appendix

From these values construct a Lineweaver-Burk plot for both experiments on the same piece of graph paper. Draw straight lines that fit best to your data. Remember that the data points far away from origo may deviate more than those closer to the y-axis. Read the Km and Vmax values for both experiments from the intercepts with the x- and y-axes.

Now answer the following questions:

1. What are the Km and Vmax (in mol*dm and mol*dm *min , respectively) of beta-galactosidase for pNP-Gal in the absence of any inhibitor?

-3 -3 -1

2. What are the Km and Vmax in the presence of inhibitor? What was the concentration of inhibitor in the reaction mixture?

3. Is the inhibition competitive or non-competitive?4. Calculate the value of kcat of beta-galactosidase for pNP-Gal (in the absence

of any inhibitor). In order to do this you need to know the concentration of the enzyme in the reaction mixture, which can be derived from its concentration in the enzyme solution (in g/l) and the molecular mass of the protein. The mass of beta-galactosidase has been estimated at 105 kDa by analytical gel filtration (Tanaka , 1975).

molar

A. oryzaeet al.

5. Calculate the value of kcat/Km for the substrate pNP-Gal. What does this value mean?

Lab report

Prepare your report according to the general guidelines in.

The report should include:

"How to write a lab report"

1. In the Abstract, which enzyme from which organism, which substrate and which inhibitor, the most important results (Km and kcat without inhibitor, Ki if calculated, how Vmax and Km changed in the experiment with inhibitor) and conclusions (which type of competition).

2. The results of the calculations above in the Results section.

3. Tables with raw data and calculated values for both experiments, either the Appendix forms filled in by hand and supplied as Appendices, or as tables in the Results section.

4. The plots of the rate of the reaction, V, vs. concentration of substrate, [S], (in M, mM or uM) for both experiments in the same diagram. Indicate the Vmax values by horisontal lines from the y-axis through the diagram. Indicate Km by vertical lines from the Km values on the x-axis up to the respective experimental curves and by horisontal lines from the y-axis at Vmax/2 to the respective experimental curves. For each experiment the two lines should intersect on the experimental curve. Remember units on both axes.

5. The Lineweaver-Burk plot for both experiments in one graph, and if



necessary, one Lineweaver-Burk plot for experiment 1 alone from which thevalues of Km and Vmax may be estimated. Remember units on both axes.

...AND DONT FORGET g/mol, mM, g/ml, nm, M-1*cm-1 etc.UNITS : m

The ambitious student may also calculate the inhibitor constant, Ki, for the inhibitor, which is a measure of how strongly the inhibitor is bound. The value of Ki corresponds to the inhibitor concentration when half of the enzyme molecules bind an inhibitor molecule (half-saturation concentration). If the inhibition is pure competitive or non-competitive, then Ki is the same as the dissociation constant, Kd, for the enzyme-inhibitor complex. The relationship between the Vmax and Km values in the absence and presence of the inhibitor are different for the two types of inhibition:

Competitive: Km(with) = Km(without)*(1+([I]/Ki))

Non-competitive: Vmax(with) = Vmax(without)/(1+([I]/Ki))

You may also look up some published data on the beta-galactosidase enzyme. In the abstract for the reference Tanaka (1975) you will find the Km value for a related substrate, oNP-Gal. Search for the article in Medline

Optional

et alhttp://

www.ncbi.nlm.nih.gov/entrez/query.fcgi

The substrate was from Sigma, cat. no. N-1252, p-nitrophenyl-beta-D-galacto-pyranoside, (1 g, price Aug 2001 SEK 330:-). FW 301 g/mol + 1 mol/mol water = 319 g/mol. For a 5 mM solution dissolve 160 mg in 100 ml 0.1 M NaAc pH 5.0. The substrate is sensitive to contamination by possible environmental beta-galactosidase and the substrate may undergo slow hydrolysis even in the absence of enzyme. May be kept a week or so in the fridge. Freeze for longer time storage.

The enzyme was from Sigma, cat. no. G-5160, beta-galactosidase from, standardised with starch (25,000 units, price Aug 2001 SEK 276:-). The

enzyme solution was prepared by dissolving 0.01 g/l powder in 0.1 M NaAc, pH 5.0. The protein content was estimated at 15% by measuring A280 and assuming an avarage protein absorbtion coefficient of 1.5 liter*g *cm . The enzyme concentration is thus given as 1.5 ug/ml. It may, however, be significantly lower since this is a crude enzyme prep, but we will use this value until an accurate determination of the beta-galactosidase content can be performed. The solution is stable at least a week in the fridge. I am not sure if the solution can be frozen.

More about the properties of beta-galactosidase and comparison of specificity with beta-gal from other organisms can be found in Zeleny (1997).

Several beta-galactosidases have high transglycosylation activity and have succesfully been applied for synthesis of oligosaccharides. A rather recent study on this is presented in Boon et al. (2000).

A very valuable source for information on enzymes is the Worthington Biochemical Corp. web site. There you can find manuals for assaying several useful enzymes and also loads of references. Unfortunately the manual for beta-galactosidase describes the enzyme which is less stable, but there are references also for the enzyme.

Supplementary information

Aspergillus oryzae

-1 -1

A. oryzaeet al.

E. coliA. oryzae http://www.worthington-

biochem.com/manual/G/BG.html



References

Boon, M.A., Janssen, A.E.M. and van 't Riet, K. (2000) Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides.

, 271-281.Enz. Microb.

Technol. 26Tanaka, Y., Kagamaiishi, A., Kiuchi, A. and Horiuchi, T. (1975) Purification and properties of beta-galactosidase from . , 241-247.Aspergillus oryzae J. Biochem. (Tokyo) 77Zeleny, R., Altmann, F., and Praznik, W. (1997) A capillary electrophoresis study on the specificity of beta-galactosidases from

and (Jack bean). , 96-101.

Aspergillus oryzae, Escherichia coli, Streptococcus pneumoniae, Canavalia ensiformis Anal. Biochem. 246


Chemicals:p-nitrophenyl-galactoside. Toxic. Avoid inhalation and

contact with skin and eyes. Use gloves.

Organisms:

Radioactivity:

Other:


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Original lab by Tom TaylorModified by Jerry Stahlberg

Page updated 2003.03.21 by [email protected] © 1998-2003. All rights reserved.Department of Molecular Biology SLU.



Table suitable for Experiment 1 / Experiment 2.

Reactionnr

Substrate-conc(M)

Background

abs(a)

Absorbancemeasured

Absorbance

(corrected)

D(b)

v(M/min)

(c) 1/S(M-1)

1/v(min/M)

1

2

3

4

5

6

7

8

9

Control

Notes

Control is the background for reaction 9. The others may be calculated as:

Background (reaction nr) = Control absorbance * [S] (reaction nr) / [S] (Control)

(a)

The reaction takes place in a 0.5 ml volume, whereas the absorbance is measured in a 1.0 ml volume. The volume corrected absorbance difference between start and stop is obtained by:

Absorbance (corrected) = 2 * (Absorbance measured - Background)

(b)

D

The reaction velocity v is obtained by :

v (M/min) = Absorbance (corrected) / (18300 * t)

(c)

D D

Back to Kinetics lab


Copyright © 1998-20002 All rights reserved.Department of Molecular Biology SLU.

2004-03-19 13:53Bke1 Biochemistry Labs

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Computer lab: Enzyme structure and function

to understand the structure and function of trypsin.

In this lab you will use SWISS PDB VIEWER (SPV) to investigate the structure and function of an enzyme. Your lab report should look a lot like the ones for the wet labs, but make sure that you include in it the answers to the questions below.

Start by downloading a pdb file for trypsin (1TPO from the Protein Data Bank).

Use SPV to load the pdb file and look at the structure. (Remember, there is a webpage with the on how to use SPV, so use it!)

Goals:

manual

1. What is the reaction catalysed by trypsin? What does the substrate look like? Are some substrates better than other substrates?

2. Look at the structure of trypsin (hide the side chains and the main chain oxygens). Guess where the active site is, zoom in on it and look at the residues there. If you can not find it by guessing, look in the book (remember chymostrypsin and trypsin are very similar).

3. Identify the catalytic triad. Describe the interactions between these residues, stating the distances between the atoms involved. A picture would help here. (Hint: first decide what the word "interaction" actually means.)

4. Now download the file for 2PTC from the PDB and load it into SPV. This is the structure of the trypsin-bovine pancreatic trypsin inhibitor (BPTI) complex. What parts of the BPTI polypeptide chain interact with trypsin? (Hint: coloring trypsin and BPTI differently will help you see this. Also, you can look at the picture of the complex in the course's Structure Gallery.) Look for specific interactions between trypsin and BPTI: give one example of a hydrogen bond, and one of a van der Waals interaction (give atom/residue names and the distances between these atoms).

5. Which residue/residues are most important in forming the substrate specificity site of trypsin? What characteristics of these residues are important? (What kind of interactions do these residues make with the inhibitor? Is size/shape important here?).

6. How does BPTI inhibit trypsin? (Is it competitive, non-competitive or uncompetitive; and why?)

7. Summarize what you have learned above, and apply it to the reaction mechanism of trypsin. Discuss how trypsin recognises its substrate and how it performs catalysis. You may find it helps to include a picture to


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support your discussion.

a) The is there for use, so - use it.

b) When looking at overall structures it is a good idea to turn off the side chains.

c) When looking at the active site it usually helps if you look only at residues that are close to the active site. For example, the program allows you to choose for display only residues within a given radius of an atom. You can use this to make a picture of with just the residues in the active site.

d) zoom in and zoom out and try to look at the structure while rotating it - it increases perception of depth.

If you run into problems, don't hesitate to ask for help.

Some tips and cluesSPV manual


[8][1] [2] [3] [4] [5] [6] [7] [9] [10] [11]

Lab by Sherry MowbrayPage updated 2001.04.04 by [email protected]

Copyright © . All rights reserved.Department of Molecular Biology SLU.


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Previous Lab index

DNA modelling tutorial

Litterature: Stryer chapters 5 and 27; Horton et al. chapters 19 and 20 - 23Materials: Push-fit molecular models from Nicholson, Labquip, England

In today’s practical you will use the same standardized system as you used for the model building of peptides/proteins. The scale is still 1 cm = 1 Å (0.1 nm) and the interatomic distances are automatically set by the depth of the socket in each unit.

Introduction

You are going to build a DNA model where you can study the configuration of the ribose ring and base pairing in detail. Each person gets a bag with units for one base pair.The ready-built base pairs will subsequently be connected to a DNA helix. To be able to do this it is important that you follow the instructions below .

Model building

EXACTLY

The colour coding is as follows:Atoms:Carbon white tetrahedrons (in the ribose-ring)Carbon grey tetrahedrons (as methyl groups)Oxygen red sphere - Note! different connection sizesPhosphorus red (yellow strip marked) tetrahedronsNitrogen light blueBases:Adenine light greenThymine dark greenUracil dark greenCytosine purpleGuanine light grey

: are to be used as hydrogen bonds within a base pairMetal pins

1. Start by building the deoxyribose rings after the scheme on the next page. You are supposed to make two identical deoxyribose units. (The structure of deoxyribose is shown in fig. 5.2 Stryer p 118; Horton page 600 fig. 19.1 shows the structure of a deoxyribonucleotide).

2. Build the bases independently and connect to each ribose.

3. Connect the bases with hydrogen-bonds, i.e. the metal-pins. (Stryer p. 122 fig. 5.12, p. 748 fig. 27.7; Horton p. 603 fig. 19.6, p. 608 fig. 19.12).

4. Time to connect all nucleotides from the group to a beautiful DNA helix. Think about the configuration of the ribose, the twist, the direction of


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the chains etc.

5. Read the bases of your DNA in triplets and translate to their corresponding amino acids. To make sure that you get directions correctly (3' or 5') you may look in Stryer p. 131 fig 5.26 (Horton p. 625 fig. 22.5) for information about the orientation of a gene and at Table 5.4 p.134 (Horton p. 657) for the genetic code.Write the answer under question 7 below.

material: 5 white tetrahedrons (one is cut), one oxygen with a narrow connection and one phosphate.

Start by connecting the oxygen at the 3' carbon (behind the plane of the paper) and continue to complete the ribose ring as shown in the figure.

It is important that the 5' carbon is connected to the bond the plane of the paper as well as later also the base (see figure).

Building of the ribose-phosphate unit

above

Questions to answer1. Which type of bonds hold the following parts of the DNA molecule

together:

the sugar-phosphate chain?the base-pairs?

2. Study the differences between ribonucleotides and deoxyribonucleotides. Why is the DNA double helix built from the latter?

3. Which are the largest differences between A-, B- and Z-DNA (Stryer p. 746-750; Horton p 612-614)?



4. You have built the "most common" form of DNA, which?

5. Now some dimensions of this DNA:How many base-pairs on one helix-turn?How long is a helix-turn in Ångstrm (1Å = 10 m)?What is the diameter of this form of DNA?

-10

6. Where are the major and minor groves located?

7. What is a propeller-twist?

8. Write down the amino acid sequence that "your" DNA is coding for!

9. What is the difference between relaxed and supercoiled DNA and why is supercoiling important?

10. What functions do the histones have in eucaryotic cells?

Previous Lab index

[11][1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

Lab by and Ulla Uhlin Margareta IngelmanPage updated 2003.05.15 by [email protected] © . All rights reserved.Department of Molecular Biology SLU.



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