Biochemistry fall 2007, lecture 4 Page 1 of 75 Plans for Today Protein Purification Electrophoresis...

Biochemistry fall 2007, lecture 4

of 75

Plans for Today

Protein Purification Electrophoresis Ultracentrifugation

Mass Spectrometry Sequencing

Quaternary structure

Why we care about structure

Structural methods Computation X-ray Crystallography

NMR Spectroscopy Cryoelectron Microscopy

Other experimental techniques


of 75

2-D Gel electrophoresis

IEF, turn 90º, then SDS-PAGE Can be used to separate ~ 200 proteins into single blobs

Allows analysis of ~all proteins from a mitochondrion or simple cell

Used semi-quantitatively to study changes in conditions(e.g. heat-shock)


of 75

2-D Gel Results

Failure to separate occurs when proteins of identical charge and nearly identical mass are present


of 75

Ultracentrifugation

Solution of macromolecules placed in rapidly spinning rotor

Heavy molecules gradually settle out

Mobility s = m(1-) / f Where m = molecular mass = inverse of particle density = solvent density f = frictional coefficient

So bigger molecules move faster, but less than proportionally: and f get bigger too


of 75

iClicker quiz! 2. A mixture containing two proteins and peptide fragments is introduced into a dialysis bag with cutoff MW = 8000. The two proteins have molecular masses of 14kDa and 20kDa. What will happen? (a) The smaller protein will remain inside and the larger one will leak out

(b) The larger protein will remain inside and the smaller one will leak out

(c) Both will remain inside and no purification will ensue

(d) Both will remain inside but the peptide fragments will leak out, resulting in partial purification

(e) None of the above


of 75

iClicker quiz, continued

3. Why is (NH4)2SO4 a more suitable salt for salting-out purifications than CaCl2? (a) Ammonium sulfate has higher solubility

(b) Calcium chloride has higher solubility

(c) Proteins have poor electrostatic affinity for chloride salts

(d) Divalent cations like Ca2+ poison proteins

(e) None of the above


of 75

iClicker quiz, continued 3. Why might it be useful to determine the structure of nitric oxide synthase? (a) To understand how Viagra works (b) To understand how the enzyme does its catalytic job

(c) To help in designing effective inhibitors of certain forms of NO synthase

(d) Both (b) and (c) (e) None of the above


of 75

iClicker quiz, concluded 4. The bacterial ribosome has a mobility of 70S. It is composed of a large component, with mobility 50S, and a small component. What would you expect the mobility of the small component to be? (a) 20S = (70S - 50S) (b) 30S, because mobility is less than proportional to mass

(c) 15S, because mobility is more than proportional to mass

(d) None of the above.


of 75

Mass spectrometry of proteins MS has been used as an analytical and preparative tool since WWII

Applications to proteins depend on availability of gentle ionization and accuracy

Two approaches available now: Electrospray MALDI


of 75

MALDI Protein dissolved in acetonitrile or EtOH in presence of crystallizable small molecule like sinapinic acid

Solvent evaporates, leaving protein adsorbed onto crystalline matrix

Laser desorbs protein ions off matrix;ions separated by MS by m/Z

Resolving power m/m > 20000


of 75

Electrospray

Protein dissolved in volatile solvent and pushed through capillary

Solvent evaporates and charged protein molecules repel one another;

Forms mist of 10 µm droplets Individual ions (“lone ions”) left behind

Lone ions subjected to MS analysis


of 75

Summary of separations: IMethod Separates by Comments

Salting out SolubilityConcentrates protein too in high salt

Dialysis molec. size removes unwanted ions

Gel Size: beads retard filtration large fastest small molecules

Ion net charge different columns exchange for + and - charges

Affinity Binding to Need to know

bound cmpds protein’s properties


of 75

Summary of separations: II

Method Separates by CommentsHPLC size: high resolution,

large fastest fast separationSDS-PAGE size (binding mobility log(mass);

to SDS) largest slowestIsoelectric Charge on can be combined focusing native protein with SDS-PAGEUltracen- sedimentation S=m(1-)/f; trifugation velocity largest fastest


of 75

Protein sequencing Usually chip away from N-terminus

Edman degradation In mild acid: Phenylisothiocyanate + protein PTH-amino acid + (rest of protein)

PTH-amino acid subjected to chromatographic analysis (elution time)

Process continues until you run out of accuracy


of 75

Quaternary structure

As we said earlier,we can have identicalchains togetheror non-identical chains

We can have mixtures, e.g. hemoglobin, which has 2 alpha chains, 2 beta chains(n.b. this has nothing to do with strands: Hb is helical)

Individual polypeptides are subunits


of 75

Why do we care about structure?

Structure defines function If we know the function already,the structure tells us how the protein accomplishes its function

If we don’t know the function, the structure may tell us what it is


of 75

How do we determine 3-D structure?

By experiment By ab initio calculation By threading By homology with existing structures

The experimental methods offer more ultimate confidence that you’re right.


of 75

When do ab initio calculations work? Small proteins Usually predominantly helical Some successes even at figuring out where the turns will go

Very limited success with sheets and coils

“Success is right around the corner”


of 75

Threading and homology modeling Predicting structure based on known structure

Threading: building fragments of known structure and weaving them through the sequence

Homology modeling: start with known structure and compute how it would change given the differences in sequence


of 75

Experimental methods Methods for complete determinations: Macromolecular crystallography Multi-dimensional NMR Cryoelectron microscopy

Methods that yield limited classes of information: UV-Vis spectroscopy Circular dichroism X-ray spectroscopy


of 75

Macromolecular crystallography

Most general and flexible tool Only atomic-resolution tool available for medium to large proteins (> 25 kDa)

Relies on diffraction, which is a form of large-scale interference among molecules organized into a regular 3-D array called a lattice

Requires crystals (not identical to solution)

Essentially static


of 75

How do we do it? Grow some crystals Requires pure protein

Sometimes fails Sometimes we get poor diffractors

Methods used to be hit-or-miss;now they’re fairly systematic

Do Experiments Get diffraction patterns

Solve phase problem Determine electron density map

Fit sequence into map

Refine structure


of 75

Diffraction experiments


of 75

Diffraction pattern

Complete dataset = 180 of these


of 75

Poll question: discuss this!

The circular stripes of scattering in the previous image are not due to the protein. What’s causing them?

(a) partially ordered water (b) fiber diffraction from the loop in which the crystal is embedded

(c) An artifact of the diffraction detector

(d) An artifact of the software used to display the image


of 75

Structures: Fourier transforms of diffraction results Position of spots tells you how big the unit cell is

Intensity tells you what the contents are We’re using electromagnetic radiation, which behaves like a wave, exp(2ik•x)

Therefore intensity Ihkl = C*|Fhkl|2

Fhkl is a complex coefficient in the Fourier transform of the electron density in the unit cell:(r) = (1/ V) hkl Fhkl exp(-2ih•r)


of 75

The phase problem

Note that we said Ihkl = C*|Fhkl|2

That means we can figure out|Fhkl| = (1/C)√Ihkl

But we can’t figure out the direction of F:Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl)

This direction angle is called a phase angle

Because we can’t get it from Ihkl, we have a problem: it’s the phase problem!

F

ab


of 75

What can we learn Electron density map + sequence we can determine the positions of all the non-H atoms in the protein—maybe!

Best resolution possible: Dmin = / 2 Often the crystal doesn’t diffract that well, so Dmin is larger—1.5Å, 2.5Å, worse

Dmin ~ 2.5Å tells us where backbone and most side-chain atoms are

Dmin ~ 1.2Å: all protein atoms, most solvent, some disordered atoms


of 75

What does this look like? Takes some experience to interpret

Automated fitting programs work pretty well with Dmin < 2.1Å

ATP binding to a protein of unknown function: S.H.Kim


of 75

How’s the field changing?

1990: all structures done by professionals

Now: many biochemists and molecular biologists are launching their own structure projects as part of broader functional studies

Fearless prediction: by 2020, crystallographers will be either technicians or methods developers

Biochemistry fall 2007, lecture 4 Page 1 of 75 Plans for Today Protein Purification Electrophoresis...

Documents

Transcript of Biochemistry fall 2007, lecture 4 Page 1 of 75 Plans for Today Protein Purification Electrophoresis...