Chapter 1

- Life characterized byo High complexityo Extraction, transformation, systematic use of energy to create and maintain

structures and do worko Sense and respond to changes in surroundingo Self-replication, enough change for evolutiono Living organisms have large # of diff compoundso Macromolecules highly specific interactionso Internal structures with defined functions

- Hierarchy of biomolecular structureo Supramolecular complexes are held together by noncovalent bondso Cell organelle supramolecular complexes macromolecules monomeric

unitso Chromatin DNA nucleotideo Plasma membrane protein amino acido Cell wall cellulose sugars

- Biochemistry: atomic composition, role of carbono Biomolecules are carbon based (>50% of cellular mass)o Common elements

H, N, O, P, So Metal ions

E.g. K, Na, Ca, Mg, Zn, Fe Play role in metabolism

o ~30 elements essential for lifeo Humans ~80% watero C (61.7%)o N (11%) o O (9.3%) o H (5.7%)

- E. Colio Water (70%)o Protein (15%)o DNA (1%)o RNA (6%)

- Biomolecules: Amino-Acids and proteinso Biomolecules are hydrocarbons with H’s replaced by functional groupso 20 common amino acids, vary by fxn group

- Biomolecules contain unique combo of fxn groupso Fxn of biomolecules depends on 3D structure

- Biochemistry is stereospecifico Stereoisomers (L and D)o Enantiomers

Non-superimposable mirror imageso Stereoisomers have different biological properties

E.g. (R)-Carvone (spearmint) vs (S)-Carvone (caraway)- Interactions between biomolecules are stereospecific

o Macromolecules have unique binding pocketso Only certain molecules fit/bind

- Biochemistry is dynamico Interactions b/w biomolecules are dynamic

- Biophysics: Quantitative description of biologyo Energy conversion

First law of thermodynamics Total energy is converted but never consumed

o Chemical/light energy ‘potential energy’ into: Kinetic energy (movement, heat) Chemical conversion – (metabolism) Macromolecular assembly – ‘loss of entropy’ Cellular work – (osmotic/electrical energy)

o ATP = chemical currency of cell- Thermodynamics: favorable vs unfavorable rxn

o Synthesis of complex molecules and many other metabolic reactions require energy (ENDERGONIC)

These rxn are thermodynamically UNFAVORABLE, (ΔG > 0) Creating order requires work/energy

o Break down of some metabolites release significant amount of energy (EXERGONIC)

E.g (ATP, NADH, NADHP) can be synthesized from sunlight/fuels Cellular concentration higher than equilibrium concentration

CELL IS NEVER AT EQUILIBRIUM – “Dyanmic Steady State”o Most of biochemistry is endergonic

- Energy Coupling: Driving Unfavorable Reactions Forward

- Kinetics: How to speed up chemical rxn?o Favorable rxn doesn’t have to occur rapidly

E.g. combustion of sugar, how do we speed it up?o Higher temperatures

Stability of macromolecules is limitingo Higher concentration of reactants

Costly, more valuable starting materials neededo Lower activation barrier by catalysis

Used by living organisms- Enzymes are protein catalysts: Function by increasing rate of rxn

o Catalysts DO NOT alter ΔGo Catalysts DO lower activation free energy of ΔG

Stabilize transition stateo Catalysis offers:

Acceleration under mild conditions High specificity Possibility for regulation

o- Enzyme and pathways

o Series of related enzymatically catalyzed reactions form a pathway

o E.g pathways Metabolic pathways – produce energy or valuable materials Signal transduction pathway – transmit information

o Provides mechanism for feedback regulation E.g. negative feedback regulation

Product of enzyme 5 inhibit enzyme 1- Genetics and evolution

o Life on earth 3.5-3.8 BYA Form self-replicating molecules

o Replication is imperfect Mutations occur

o Mutations give organisms advantage in given environment likely to be propagated

Chapter 2

- Non-covalent interactionso Dipole interactions and H-bonds

Electrostatic interactions b/w uncharged polar moleculeso Hydrogen bonds

b/w neutral groups or peptide bondso Ionic (Coulombic) interactions

Electrostatic interactions b/w permanent charged species or b/w ion and permanent dipole

Attraction or repulsiono Hydrophobic effect

Ordering of water molecules around non-polar substanceso Van der Waals interactions

Weak interactions b/w all atoms Attractive (dispersion) and repulsive (steric) component Any 2 atoms in close proximity

- Van der Waals interactions

oo 2 components

Attractive (London force) Depend on polarizability

Repulsive force (steric repulsion) Depend on size of atom

o Attraction/repulsion Attraction dominates longer distance

0.4-0.7nm (4-7 angstroms)o 1 ANGSTROM = 0.1 nm! Or 10-10 m

Repulsion dominates at very short distances There is a Minimum optimal energy distance

Van der Waals contact distance- Van der Waals interactions: Biological importance

o Universal b/w any 2 atoms near ea/o

o Weak individually Easily broken, allow for reversible rxn

o Importance Determines steric complementarity

E.g. macromolecular interactions ‘shape complementarity’ Stabilizes biological macromolecules (stacking in DNA) Facilitate binding of polarizable ligands

- Hydrogen bonds: biological importanceo Source of unique properties of watero Structure and fxn of biopolymers

Proteins, nucleic acids, polysaccharides, lipids, …o Binding of a substrate to proteins

E.g. binding hormones to receptors, substrates to enzymeso Matching of DNA, mRNA, tRNA

- Hydrogen bonds: strong dipole-dipole interactiono Typically involve 2 electronegative atoms e.g. Nitrogen and Oxygen

o Typically 4-6 kJ/mol for bonds with neutral atoms, 6-10 kJ/mol for bonds with 1 charged atom

o Strength of H-bond depend on distance and orientation Ideally 3 atoms involved in a line

- Hydrogen bonds: biological importanceo B/w hydroxyl group of an alcohol and watero b/w carbonyl group of ketone and watero b/w peptide groups in polypeptideso b/w complementary bases of DNA

- Water and hydrogen bondso Water can serve as both H-Doner and H-Acceptor

Up to 4 H-bonds per water moleculeo Gives water its anomalous properties

High boiling point High melting point Large surface tension

o H-bonds are weak (20 kJ/mol)- Water as a solvent

o Water is a good solvent for charged and polar substances Amino acids and peptides Small alcohols Carbohydrates

o Water is poor solvent for nonpolar substances Nonpolar gases Aromatic moieties Aliphatic chains

o Some polar biomolecules Glucose, Glycine, Aspartate, Lactate, Glycerol

o Some nonpolar biomolecules Wax, amphipatic phenylalanine, phosphatidylcholine

- Water as a solvent: Ionic molecules – Saltso Highly dielectric constant reduces attraction b/w oppositely charged ions in salt

crystal Almost no attraction at large (>40 nm) distance

o Strong electrostatic interactions b/w solvated ions and water lowers energy of system

o Entropy increases Ordered crystal is dissolved

- Water as a solvent: Gas molecules

o Polar gases dissolve better than nonpolar gases E.g.

Polar: Ammonia, Hydrogen sulfide Nonpolar: N, O, CO2

o Nonpolar gases chemically converted, or bound to other molecules for biological transport

E.g. myoglobin- Water as a solvent: Hydrophobic molecules

o Hydrophobic molecules poorly dissolved in water explained by entropyo Bulky water has little order

High entropyo Water near hydrophobic solute highly ordered

Low entropyo Low entropy thermodynamically unfavorable

Hydrophobic solutes have low solubilityo Hydrophobic effect minimizes loss of water entropy

- Water as solvent “hydrophobic effect”o Association or folding of nonpolar molecules in aq solutiono Is one of the main factors behind

Protein folding Protein-protein association Formation of lipid micelles and membranes Binding of steroid hormones to their receptors

o Does NOT arise b/c of direct attractive force b/w 2 non-polar molecules In fact, ΔH > 0 for transfer of many nonpolars into benzene from water;

while ΔG < 0 (so dominated by TΔS)o Origins of hydrophobic effect

Consider amphipathic lipids in water Lipid molecules disperse in solution

Nonpolar tail is surrounded by ordered water molecules Entropy of system decreases System is now unfavorable

o Nonpolar chains of the amphipathic molecule aggregate Fewer water molecules are ordered Released water molecules will be more random, entropy increases

o Aggregation continues Only polar “head groups” of ampipathic molecules are exposed Make energetically favorable H-bonds with water molecules

- Water and protein-ligand interactions: H-bondingo Water is a ligand

- Water and osmotic pressureo Cell is surrounded by a semi-permeable membrane

Water is conducted through specialize protein called Aquaporins Cell in Isotonic = no net water movement Cell in hypertonic = water out, cell shrinks Cell in hypotonic = water in, burst

- Osmotic dysfunction and human diseaseo Ocular – cataracts, glaucomao Kidney – hypertension, diabetes insipidiso CNS – neuromyelitis opticao Skin – eczema, wound healing

- Ionization of water

o OH bonds are polar and can dissociate Products are proton H+ and hydroxide ion OH- Dissociation of water is rapid, reversible process

o Most water molecules remain unionized, thus pure water has low electrical conductivity

o Equilibrium strongly to lefto Extent of dissociation depend on temperature

- Proton hydrationo Protons do not exist free in solution

They are hydrated forming hydronium H3O+ ionso Hydronium ions are solvated by nearby water moleculeso Covalent and hydrogen bonds are interchangeable

Allows for fast mobility of protons in water via proton hopping- Water ionization: quantitative treatment

o Concentrations of species involved in chemical equation described by the equilibrium constant

Keq = [H+]*[OH-]/[H2O]o Keq can be experimentally determined = 1.8*10-16 M at 25°Co [] = concentration Molarity, M, mol/Litero [H2O] = 55.5M determined from water density @ 25°C

(1000 g/L) / 18 (g/mol) = 55.5 M

o- The pH scale

o Way to express [H+]o In neutral solution [H+] = [OH-] and pH = 7o pH and pOH always add to 14o pH can be negative ([H+] > 1 M)o What is pH?

pH = -log [H+] –log[10-7] = 7 Kw = [H+][OH-] = 1*10-14 M2

–log[H+] – log[OH-] = +14 pH + pOH = 14

o pH depends little on H+ from watero pH altered by presence of other acids and bases that inc or decr [H+]

Acid release proton [H+] in water Bases accept proton [H+]

o Acid dissociation in water HA H+ + A-

Conjugate pairs

- Weak acids/bases and dissociation constant

oo Biological acids/bases are often weako Weak electrolyte dissociate only partially in watero Extent of dissociation is determined by acid dissociation constant Kao Can calculate pH if Ka is known

o

oo Large Ka = greater dissociation = STRONGER ACIDo Lactic acid has Ka = 1.4 x 10-4

o [Lactic acid] in blood ~1 mMo Assuming no other buffers are present, what is the pH?

- pKa values – measure of acidityo pKa convenient way to express Ka

pKa = -logKa (strong acid large Ka small pKa) DON’T CONFUSE WITH pH

pKa is constant and intrinsic property of the moleculeo Most biological buffers have pKa between 2-13

- Bufferso What is a buffer?

Mixture of weak acid and its Anion (conjugate base) Buffers RESIST CHANGE in pH When pH = pKa – there is a 50:50 mixture of acid and anion forms Buffering capacity of weak acid/base system is greatest at pH = pKa Buffering capacity is lost when the pH differs from pKa by more than 1

pH unit Cell is buffered by a variety of weak acids/bases

- Buffers and pKao We can determine the pKa of a weak acid/base experimentally by producing

titration curveo Systematic addition of strong base of known concentration while measuring the

pHo The titration curve of acetic acid

pH is recorded at each addition of OH- OH- is recorded as a fraction required to fully deprotonate the acid

“equivalents” At midpoint [HA] = [A-], and pH = pKa The blue shaded area is the pH region where the HA/A- is a good buffer

o Weak acids have unique pKa’so In the lab

Buffer systems are chosen for buffering capacity over a desired pH range Proteins and nucleic acids are stable over a narrow pH range Enzymes catalyze reactions at an optimal pH

- Henderson-Hasselbalch Equationo We can quantitatively describe the titration curve of a weak acid using the HH-

equation

o The HH equation

Quantitatively describes the shape of the titration curve Determine pKa, given pH and molar ratio of HA/A- Determine pH, given pKa and molar ratio of HA/A- Determine ratio of HA/A- given pH and pKa

- Proteins and other Biomoleculeso Amino acids are weak acid/bases

Useful to know their protonation state

- Henderson-Hasselbalch Equation

oo Determine the fraction of histidine group that is protonated at pH = 7.3

pK1 = 1.8 (carboxyl group) pK2 = 6.0 (imidazole group) pK3 = 9.2 (amino group)

- Ratio vs Percentage

o- HH Equation

oo The cell cytoplasm is maintained at pH~7.4o Under normal conditions, lactic acid is at a concentration of 1mM

What are the concentrations of the acid and base forms?

- Biological buffer systemso Maintenance of intracellular pH is vital to all cells

Enzyme catalyzed rxn have optimal pH Solubility of polar molecules depends on H-bond donors and acceptors Equilibrium b/w CO2 gas and dissolved HCO3

- depends on pHo Buffer systems in vivo are mainly based on

Phosphate, concentration in millimolar range Histidine, efficient buffer at neutral pH Bicarbonate, important for blood plasma

o Important aspects of Drug Design/Pharmacokinetics Drugs are exposed to various pH conditions in the body

Can change their function/recognition by substrate Ionized molecules do not cross cell membranes easily

- Water as a Reactanto Water is not just a solvent and medium for biology – it plays an active role in

biochemistry Acid base chemistry Another important example, conversion of CO2 to Carbonic acid

o Condensation/Hydrolysis Rxns

oo ATP made from energy from proton gradients across membranes of chloroplasts

and mitochondriao Other condensation reactions

Polymerization of amino acids, nucleic acids, carbohydrates ALL THESE ARE ENDERGONIC – REQUIRE HYDROLYSIS

OF ATPo Oxidation/Reduction Rxns

o

Chapter 3: Amino Acids, Peptides and Proteins

- Proteins: Main agents of biological functiono Enzymatic Catalysis

Enolase (in glycolytic pathway) DNA polymerase (in DNA replication)

o Transport Hemoglobin (transport O2 in blood) Lactose permease (transports lactose across cell membrane)

o Structure Collagen (connective tissue) Keratin (hair, nails, feathers, horns)

o Motion Myosin (muscle tissue) Actin (muscle tissue, cell motility)

- Amino Acids: Building blocks of proteinso Proteins are linear heteropolymers of alpha-amino acidso Amino acids have properties that are well-suited to carry out a variety of biological

functions Capacity to form stable polymers Useful acid-base properties Varied physical properties Varied chemical functionality

- Amino Acids: Atom namingo Organic nomeclature vs Biochemical designation

Start from one end and number down the carbons Start from alpha-carbon and go down the R-group

Only concered with biochemical nomenclature

- Amino acids: Amino acids are Chiral (EXCEPT GLYCINE)o The α-carbon has always 4 substituents and is tetrahedralo All have an acidic carbonyl group, basic amino group, and an alpha-hydrogen

connected to the α-carbono Each amino acid has a unique substituent Ro In glycine 4th substituent is (also) hydrogen

o L- and D- notations are in comparison to Glyceraldehyde example below L-Levorotary

Left handed rotation of polarized light Highest priority group on the LEFT

Proteins ONLY contain L-amino acids

- Amino Acid Classificationo 20 common amino acidso 5 basic groups depending on R-groups

Nonpolar, aliphatic (7) Aromatic (3) Polar, uncharged (5) (+) charged (3) (-) charged (2)

- Nonpolar aliphatic R-groupso G, A, P, V, L, I, M

- Aromatic R-groupso F, Y, W

These AA side chains absorb UV light at 270-280 nm- Polar, uncharged R groups

o S, T, C, N, Q Cysteines can form disulfide bonds ‘covalent bonds’

- (+) charged R-groupso L, R, H

H, pKa ~6.0- (-) charged R-groups

o D, E- Uncommon Amino Acids

o Arise by POST-TRANSLATIONAL MODIFICATIONS of proteinso Reversible modifications, especially phosphorylation, are important in regulation and

signalingo Otherwise are important products/intermediates in metabolic pathwayso There are also “un-natural” (synthetic) AA that can be tricked into protein structures

Used to probe function of a protein, or introduce new functionalities- Uncommon/modified Amino Acids (examples)

o Phosphorylation

o Acetylation

o Methylation

o Adenylation

- Ionization of Amino Acidso At acidic pH (FULLY PROTONATED)

Carboxyl group is protonated and the amino acid is in the CATIONIC formo At Neutral pH (ZWITTERION FORM)

Carboxyl group is DEPROTONATED but the AMINO group is PROTONATED

Net charge is 0, these ions are called Zwitterionso At alkaline pH (FULLY DEPROTONATED)

The amino group is neutral (-NH2) and the amino acid is in the ANIONIC form

o Amino acids with uncharged side chains, e.g. GLYCINE, have 2 pKa valueso For glycine

pKa of α-carboxyl = 2.34 pka of α-amino = 9.6 It can act as a buffer in two pH regimes

oo Zwitterions predominate at pH values b/w the pKa values of amino and carboxyl

group

for amino acid without ionizable side chains, the ISOELECTRIC POINT (equivalence point, pI) is

pI = (pK1 + pK2) / 2 At the pI, net charge = 0

Least soluble point in water Does not migrate in electric field

o The chemical environment affects pKa values: Α-carboxy group is much more acidic than in carboxylic acids Α-amino group is slightly less basic than in amines

o Some Amino acids with ionizable side chains, such as the charged amino acids, have 3 pKa values

o Ionizable side chains can be titrated Titration curves are more complex pKa values are discernable if two pKa values are more than 2 pH units apart

o How to calculate the pI of an amino group with an Ionizable R-group? Identify the net zero charge species Identify pKa value that defines the acid strength of this zwitterion: (For Glu =

pKa1) Identify pKa value that defines the base strength for this zwitterion (For Glu =

pKR) Take the average of these two pKa values (For Glu = 3.22)

o Example What is the pI of histidine?

Pk1 = 1.82 pKR = 6.0 pK2 = 9.17

(9.17 + 6)/2 = pI

pI = 7.59 MUST KNOW HOW TO DRAW TITRATION CURVE FOR GIVEN

AMINO ACID AND LABEL THE PK’s and pI!

- Peptide Formation: Peptide bondo Peptide bond:

Formed by condensation reaction b/w 2 aa

- Naming and conventiono Peptides are small condensation products of aa’s

“small” compared to proteins (Mw < 10kDa)o Polypeptide chain

o Always listed in NC direction- Peptide naming/convention

o Numbering/naming starts from amino terminus 3 letter code

Written as Ser1-Gly2-Tyr3-Ala4-Leu5 One letter code

SGYAL Should be able to recognize amino acid structure and name them using both

conventions- Peptide functions

o Hormones and pheromones Insulin (think sugar) Oxytocin (think childbirth)

Sex-peptide (think fruit fly mating)o Neuropeptides

Substance P (pain mediator)o Antibiotics

Polymyxin B (for Gram – bacteria) Bacitracin (for Gram + bacteria)

o Protection (e.g. toxins) Apamin (bee venom) Conotoxin (cone snails) Chlorotoxin (scorpions)

- Peptide formation: peptide bondo Formed by condensation rxn b/w 2 aao Endergonic rxn

Require ‘activation’ by chemical modification of N or C terminus In cells, c terminus is activated by ATP For chemical synthesis we use other molecules for activation

- Peptide Synthesiso Use solid-state Fmoc chemistryo Solution state chemistry is too difficult to purify each step of rxno Fmoc is a protecting group! – NOT AN ACTIVATING GROUP

- Fmoc peptide syntehsiso Stepwise synthesis carried out in C to N directiono 1. Residue 1

Fmoc protected N-terminal attached to styrene beado 2. Remove Fmoc with mild baseo 3. Residue n+1

With DCC activated C-terminal added and peptide bond formedo 4. Repeato 5. Removal of peptide from bead using TFA

- Peptide formation: chemical synthesiso Peptides over 100aa are not practical for synthesis

oo Ribosome can synthesize 100aa in 5 seconds (error rate 1/10,000)o Amino acid activated by ATP and ribosome catalysis

- Polypeptides (covalently linked a-amino acids and possibly:o Cofactors

general term for functional non-amino acid component Metal ions, organic molecules

o Coenzymes Used to designate organic cofactors of enzymes

NAD+ in lactate hydrogenaseo Prosthetic groups

Covalently attached cofactors Heme in myoglobin

o Other modifications- Proteins size and composition

o Simple proteins contain only amino acidso Conjugated proteins contain prosthetic groups:

Lipids Sugars Metals Cofactors

oo Peptide vs Protein (latter are typicaly ~10,000 Da)

oo Proteins vary in their amino acid compositiono The molecular weight of a simple protein estimated by # of AA it contains

(# of AA) x 110 Da = ~Molecular weight Note:

Average amino acid mass is 138 Da However, small amino acids are more common The average Mw of amino acids used by proteins is = 128 Da In a polypeptide, H2O is removed due to peptide bond, so average mw

is reduced to 128-18 = 110 Da per amino acid Peptides and proteins, What we trying to learn?

Sequence and composition?o How vary b/w individuals and species?o How related to other proteins?

What is 3d structure?o How related to fxn?o How did it find native fold

How achieve its biochemical role?o How is its biochem role disrupted in disease?

Where is it localized within cello How does it co-localize with other proteins/organelles?

What are its physico-chemical properties?o How do they relate to its fxn?o How can we exploit them for analysis?

- Amino acid classification: Aromatic R-groups and UV absorptiono F, Y, W

These aa side chains absorb UV light at 270-280 nm Can be Used for detection and quantification

o Spectroscopic detection of proteins/polypeptideso Tryptophan and Tyrosine are the strongest

o UV absorbance maxima ~275-280 nmo Concentration ca be determined using spectrophotometry and Beers law

A = ε·c· l A = absorbance = -log (l/lo) Ε = extinction coeff C = concentration l = path length

E.g. Purified M-E-T-S-Y-Q-N-L-L-W-T-W-S-G-G-K Extinction coeff given

o Tyr = 1280 M-1*cm-1

o Trp = 5690 M-1*cm-1

What is the overall E for peptide?o 1280 + (2x 5690) = 12660 M-1*cm-1

Given A = 1.23 Given 1cm cuvette What is peptide concentration?

o (1.23)/(12660*1cm) = 9.72*10-5 M- Peptides and Proteins, Separating from a mixture (purification)

o Separation relies on differences in physical and chemical properties Charge Size Affinity for ligand Solubility Hydrophobicity Thermal stability

o Common techniques for preparative separation Centrifugation

Separation based on density (i.e. differential centrifugation) Precipitation (followed by centrifugation/filtration)

Separation based on solubility in the presence of various chemical and/or thermal conditions (e.g. salts, pH, organic solvents, temp. etc.)

Chromatography Separation based on interaction with solid-phase matrix

o E.g. charge, hydrophobicity, size, interaction with ligand Most protein purification strategies utilize a combination of these approaches! Differential centrifugation

Tissue homogenization (tissue homogenate) Low speed centrifugation 1000 g, 10 min

o Pellet contains whole cells, nuclei, cytoskeletons, plasma membranes

Supernatant subjected to Medium speed centrifugation 20,000 g 20mino Pellet contains mitochondria, lysosomes, peroxisomes

Supernatant subjected to high speed centriguation 80,000 g, 1ho Pellet contains microsomes(fragments of ER), small vesicles

Subject to very high speed centrifugation (150,000 g 3h)o Supernatant contains soluble proteinso Pellet contains ribosomes, large macromolecules

- Peptides and proteins, separating from a mixture (purification)o Column Chromatography

Mobile phase – protein solution Stationary phase – porous matrix Proteins in mobile phase interact with the stationary phase matrix based on

unique physico-chemical propertieso Ion-exchange chromatography

Separation based on the surface charge characteristics of the protein/biomolecule

Cation exchange – negatively charged resin Choose if buffer pH < pI (positively charged)

Anion exchange – positively charged resin Choose if buffer pH > pI (negatively charged)

Strongly bound proteins are eluted with high salt buffer Method is selected based on the protein’s isoelectric point pI How it works:

Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier

Example, protein of interest has pI = 8.9, buffer pH = 7 What ion exchange method would you choose for purifaction? First decide – is the protein + or – charged at pH = 7.4? Buffer pH is below pI, so positively charged Select a cation-exchange column

o Size-exclusion chromatography AKA Gel-filtration Separation based on size of protein/biomolecule

Larger proteins flow through faster than smaller ones Many different pore sizes available

Selected based on expected MW of the protein May also be used to determine the MW of your protein by comparing

a set of calibrated standards How it works:

protein mixture is added to column containing cross-linked polymers protein molecules separate by size; larger molecules pass more freely

appearing in the earlier fractions Example:

You have protein of 376 aa

You have choice b/w S75 or S200 SEC columno Note: S75 is for proteins (10-100 kDa)o S200 is for proteins (100-1000 kDa)

Which column would you use? (376 AA) x (110 Da) = 41,360 Da

o Use the S75 columno Affinity chromatography

Separation based on affinity to a ligand Variety of ligands

Small molecule Metal Antibody/protein

Affinity ‘tags’ can be genetically engineered into a protein target These methods can be very specific

How it works: Solution of ligand is added to column Protein mixture is added to column containing polymer bound ligand

specific for protein of interest Unwanted proteins are washed through column Proteins of interest are eluted by ligand solution

- Peptides and proteins, assessing the quality of purificationo Enzymatic analysis

Total activity vs specific activity Enzymatic activity

o 1 Unit = transforms 1 umol of product per min at 25C Specific activity

o = total activity/total protein During purification total activity should not change much; specific activity

should get higher

For non-enzymes require a different assay

o General assay to assess protein purity Gel Electrophoresis

Electric field pulls proteins according to their charge Gel matrix hinders mobility of proteins according to their size and

shapeo Smaller proteins migrate faster than larger ones

o red one is cathode (+)o Polyacrylamide Gel Electrophoresis (PAGE)

Separates biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.

o Denaturing Electrophoresis (SDS-PAGE) SDS – sodium dodecyl sulfate – a detergent

SDS micelles bind and unfolds ‘denatures’ the proteins

Gives all proteins a uniformly negative charge Gives all proteins a uniform shape ‘linear polypeptide’ Rate of movement will only depend on linear size: small proteins will

move faster Typically incorporates a ‘reducing agent’

o E.g. B-mercaptoethanol (BME) or Dithiothreitol (DTT)

These are used to reduce disulfide bonds

- Assessing quality of purificationo Denaturing electrophoresis ‘SDS-PAGE’o Typically incorporates reducing agent

E.g. B-Mercaptoethanol (BME) or Dithiothreitol (DTT) These reagents used to reduce disulfide bonds

- Analyzing physico-chemical properties

o

oo

o- Protein sequencing

o Primary AA sequence provides Protein ID Insight into physico-chemical properties (MW + pI) Insight into 2°, 3° structure (sometimes 4° structure) Insight into biological function, disease and evolutionary history

Actual sequence generally determined from DNA sequence Edman Degradation (Classical method)

Successive rounds of N-terminal modification, cleavage, and identification

Can be used to identify protein with known sequence Mass Spectroscopy (modern method)

MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the aa sequence

Can e used to determine PTM- Protein Sequencing (EDMAN DEGRADATION)

oo Benefits

Only require 10-100 pico-mole of proteino Drawbacks

Can only sequence ~30 AAo How to overcome drawback?

Cleave larger proteins into smaller pieces first E.g. using proteases, or chemical cleavage

- Protein Sequencing – Protease Enzymeso

- Protein Sequencing – Mass Spectroscopyo Modern methods of protein mass and sequence analysis done by Mass-Spec

oo Electrospray Ionization Mass Spectroscopy (ESI-MS)o

o

o

o

o- DNA Sequence analysis

o

- Primary Sequence Analysis

o

o

o

o

Chapter 4

- 3D structure of proteinso Major themes

3D structure determined by 1° sequence Protein fxn depend on structure “Form & Function” Most proteins exist in one or a few # of stable structures Protein structure is primarily stabilized by non-covalent interactions Proteins may share common structural features Protein structures are not static – Dynamics is important to function, and some

are completely unstructured- Overview of Protein Structure

o Unlike most organic polymers, protein molecules adopt a specific 3D conformation

o This structure is able to fulfill a specific biological functiono This structure is called NATIVE FOLD or NATIVE CONFORMATION

- 4 levels of protein structure

o- Overview of Protein Structure

o Native fold is stabilized by a large # of favorable interactions w/I protein Typically, this is the THERMODYNAMICALLY MOST STABLE There is a COST IN CONFORMATIONAL ENTROPY of folding the protein

into one specific native fold Proteins MAY HAVE MORE THAN ONE STABLE CONFORMATION

Important to catalysis (e.g. binding and release of substrate)- Non-covalent interactions are maximized

o Hydrophobic effect (Major component) Release of water molecules from the structured solvation layer around the

molecule as protein folds increase the net ENTROPYo Hydrogen bonds

Interactions of N-H and C=O of the peptide bond leads to local regular structures such as a-helices and B-sheets

o London dispersion Medium-range weak attraction b/w all atoms contribute significantly to the

stability in interior of proteino Electrostatic interactions

Long-range strong interactions b/w permanently charged groups Salt-bridges, esp. buried in hydrophobic environment strongly stabilize the

protein- Structure of the peptide

o Protein structure partially dictated by properties of the peptide bondo Peptide bond is a resonance hybrid of 2 canonical structures

o Resonance causes peptide bonds To be less reactive (compared to esters for example) To be rigid and nearly planar To exhibit large dipole moment in favored trans configuration

- Phi/Psi Dihedral Angleso Rotation around the peptide bond is not permittedo Rotation of bonds connected to the a-cabon is permitted

o

o

- Protein Secondary Structureo Secondary structure refers to local spatial arrangement of polypeptide backboneo 2 regular arrangements are common

a-helix stabilized by H-bonds b/w residues nearby in sequence

B-sheet Stabilized by H-bonds b/w adjacent segments that may not be nearby

in sequenceo Irregular arrangement of polypeptide chains is called RANDOM COIL

- Right handed a-helixo 3.6 residues (5.4Å) per turno Stabilized by H-bonds b/w nearby backbone amides

(n and n+3 residues)o Polypeptide bonds are aligned roughly parallel with helical axiso Side chains point out roughly perpendicular w/ helical axis

- The Alpha Helix-

o Inner diameter of the helix (no side chains) is about 4-5Å Too small for anything to fit “inside”

o The outer diameter of the helix (with side chains) is 10-12Åo Residue 1 and 8 align nicely on top of each othero Not all polypeptide sequences adopt a-helix structureso Small hydrophobic residues Ala and Leu are strong helix formerso Pro acts as a helix breaker because the rotation around N-Ca bond is impossibleo Gly acts as helical breaker because the tiny R-group supports other conformationso Attractive or repulsive interactions b/w side chains 3-4 amino acids apart will affect

formation

o- The Helix Dipole

o Recall that the peptide bond has a strong dipole moment Carbonyl O = negative Amide H = positive

o All peptide bonds in the a-helix have a similar orientationo Result in a large macroscopic dipole moment

Negatively charged residues often occur near the positive end of the helix dipole

- Beta-sheet

o The backbone is in a more extended conformation

o The pleated sheet-like arrangement of backbone is held together by hydrogen bonds b/w the backbone amides in different strands

o Side chains protrude from the sheet alternating in up and down direction

oo Parallel or Antiparallel orientation of two chains w/I a sheet are possibleo Parallel B-sheets

H-bonded strands run in the SAME DIRECTION Resulting in bent H-bonds (weaker)

o Antiparallel B-sheets H-bonded strands run in the OPPOSITE DIRECTIONS Resulting in linear H-bonds (stronger)

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- Beta-turnso B-turns occur frequently whenever strands in B-sheets change the direction

The 180° turn is accomplished over 4 amino acidso The turn is stabilized by H-bond from carbonyl oxygen to amide proton at the n+3

positiono Proline in position 2 or Glycine in position 3 are common in B-turns

o- Proline Isomers (Cis and Trans)

o Most peptide bonds not involving proline are in the trans configuration (<99.95(o For peptide bonds involving proline, about 6% are in the cis configuration. Most of

this 6% involves B-turnso Proline isomerization is catalyzed by proline isomerases

o- Analysis of Secondary Structure Circular Dichroism (CD)

o Spectroscopic technique to quantitatively assess secondary structure contento CD measures the molar absorption differences Δε or left- and right-circularly

polarized light Δε = εL – εR

Chromophores in a chiral environment produce characteristic signals CD signals from peptide bonds depend on the chain conformation

o- Protein Tertiary Structure

o Tertiary structure Overall spatial arrangement of atoms in a polypeptide chain or in a protein

o 3 major classes Globular proteins

Water-soluble proteins Lipid-soluble membranous proteins

Fibrous proteins Typically insoluble; made from a single secondary structure

Intrinsically disordered proteins Lack stabile/ordered structure

o Found with a huge variety of shapes and sizes but some general features are shared Compact fold Hydrophobics concentrated at the interior of hydrophilics at surface Stabilized by many H-bonds and ion-pairs

o While overall structures are often very complex, most proteins are assembled by linking together common structural features “motifs” or “folds”

- Protein Motifs (Folds)o Motif/Fold – recognizable folding pattern of 2 or more connected 2ndary structural

elementso All alpha, All beta, or Mixed alpa/betao Motifs can be found as reoccurring structures in numerous proteins

o Most proteins are made of different motifs folded together

oo Specific connectivity b/w sequential 2ndary elements are favored

oo Larger and more complex folds may be constructed from smaller simpler motifs

oo Hierarchy

Class: all α, all β, α/β, and α+β Each class has many folds

Some are very common, some are unique to only 1 known protein

o Structural classification of proteins (SCOP) database

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- Protein Domainso Domain – part of a protein that is independently stable

Multiple domains may have independent functions within the entire protein May be separated by a linker (independent), or closely packed with eachother

- Protein Quaternary Structure

o Quaternary Structure – formed by spontaneous assembly of individual polypeptides into a larger functional oligomer

o e.g. Hemoglobin – Functional Unit is a Tetramer- Protein Structure Database (PDB)

o There are 20-25,000 protein coding regions in the human genomeo Only ~1,400 unique folds in nature

- Globular Protein Structureo Myoglobin

First atomic model of a proteino Features

Hydrophobic core Hydrophilic surface Shape is adapted to function

o Group of enzymes involved in glycolysis Even w/i the small group of related function, you can observe a variety of

size, shape, symmetry, oligomeric state, etc.- Globular Protein Structure and Function

o Globular Proteins Most common in biologyo Perform variety of biological functions

Enzymes Transporters Regulatory proteins Antibodies

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o Signal transduction nearly half- Fibrous Protein Structure and Function

o Adapted for Structural Roleso Simple repeating element of secondary structureo Insoluble in watero Hydrophobic surfaces of similar peptides pack together = fibrilso

- Fibrous Protein Quaternary Structure

o- Fibrous Protein Structure

o Collagen Gly and Pro-rich left handed helix Three chains form a right handed triple helix Higher tensile strength than steel wire Gly residues facilitate fibril packing Many triple helices assemble into a collagen fibril Stabilized by inter-strand H-bonding and crosslinking b/w modified lysines

(unusual)

o 4-Hydroxyproline in Collagen

Forces the proline into a favorable pucker conformation Offer more hydrogen bonds b/w 3 strands of collagen The post-translational processing is catalyzed by prolyl-hydroxylase and

requires: a-ketoglutarate, oxygen and ascorbate (vitamin C) Lack of vitamin C can cause scurvy (degeneration of connective tissue)

oo Example of B-sheet fibrous protein: Silk Fibroin

Main protein in silk from moths and spiders Anti-parallel B-sheet structure Small side chains (Ala- and Gly) allow the close packing Structure is stabilized by H-bonding w/i sheets London dispersion interactions b/w sheets

Extremely strong material Strong than steel Can stretch a lot before breaking

- Intrinsically Disordered Protein Structureo Proteins, or protein segments that lack definable structureo Composed of amino acids whose higher concentration forces less-defined structure

Lys, Arg, Glu and Proo Functions include

Protein/protein interactions Can adopt many different conformations, facilitating highly specific

interactions with several partner proteins Cell signaling

Common sites of post-translational modification (e.g. phosphorylation) involved in signaling pathways

Domain Tethering Linking together functional domains into a single molecule or

oligomeric complexo Phosphorylated Kinase Inducible Domain (pKID)

Protein interaction induces secondary structure Allows for intimate binding interactions

o P53 protein Several different conformations possible Allows for multiple binding partners

o Protein Kinase A Tethering of Functionally Coupled Proteins/Domains

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- Methods of determining protein structureo 3 major methods

X-ray crystallography NMR spectroscopy Electro microscopy

- Determining Protein Structure (X-ray Crystallography)o X-ray crystallography is by far the most common and successful technique

Diffraction technique: uses coherent X-ray light sourceo Required steps:

Purify the protein (mg quantities) Crystalize the protein Collect diffraction data Calculate electron density map Build model into density

o Pros No size limits Well-established High resolution

o Cons Crystallization can be difficult/impossible

Especially for membrane proteins- Determining Protein Structure (Biomolecular NMR Spectroscopy)

o NMR Spectroscopy NMR active nuclei (e.g. 1H , 13C) absorb radio frequencies when placed in a

strong magnetic field

o Required steps Isotope labeling Purify protein (mg quantities) Collect NMR data Assign NMR spectra Calculate structure

o Pros Does not require crystals Information on structure and dynamics

o Cons Difficult for insoluble membrane proteins Difficult for larger proteins (>50 kDa)

- Determining Protein Structure (Biomolecular CryoEM)o Electron Cryo-Microscopy (cryoEM)

Imaging technique using electron beam as light sourceo Required steps

Purifying the protein (ug quantities) Collect image data Calculate 3d reconstruction Build model into density map

o Pros Does not require crystals No size limit (except for small proteins)

o Cons Technically difficult Best for large proteins (>200 kDa)