protein methods andy howard introductory biochemistry fall 2010, iit

Protein MethodsProtein Methods

Andy Howard

Introductory BiochemistryFall 2010, IIT

09/09/2010 Protein Methods and Function p. 2 of 62

Proteins are worth studyingProteins are worth studying

We’ll perform a quick overview of methods of studying proteins– Purification methods– Analytical methods– Structural methods


The Protein Data BankThe Protein Data Bankhttp://www.rcsb.org/This is an electronic repository

for three-dimensional structural information of polypeptides and polynucleotides


What it containsWhat it contains

68000 structures as of September 2010– Most are determined by X-ray

crystallography– Smaller number are high-field NMR

structures– A few calculated structures, most of which

are either close relatives of experimental structures or else they’re small, all-alpha-helical proteins


What you can do with the PDBWhat you can do with the PDB

Display structures Look up specific coordinates Run clever software that compares

and synthesizes the knowledge contained there

Use it as a source for determining additional structures


Protein PurificationProtein Purification

Why do we purify proteins?– To get a basic idea of function we need

to see a protein in isolation from its environment

– That necessitates purification– An instance of reductionist science

Full characterization requires a knowledge of the protein’s action in context


Salting OutSalting Out Most proteins are less soluble in high salt

than in low salt In high salt, water molecules are too busy

interacting with the primary solute (salt) to pay much attention to the secondary solute (protein)

Various proteins differ in the degree to which their solubility disappears as [salt] goes up

We can separate proteins by their differential solubility in high salt.


How to do itHow to do it

Dissolve protein mixture in highly soluble salt like Li2SO4, (NH4)2SO4, NaCl

Increase [salt] until some proteins precipitate and others don’t

You may be able to recover both:– The supernatant (get rid of salt; move on)– The pellet (redissolve, desalt, move on)

Typical salt concentrations > 1M


DialysisDialysis

Some plastics allow molecules to pass through if and only ifMW < Cutoff

Protein will stayinside bag, smaller proteins will leave

Non-protein impurities may leave too.


Gel-filtration chromatographyGel-filtration chromatography

Pass a protein solution through a bead-containing medium at low pressure

Beads retard small molecules Beads don’t retard bigger molecules Can be used to separate proteins of

significantly different sizes Suitable for preparative work


Ion-exchange Ion-exchange chromatographychromatography

Charged species affixed to column

Phosphonates (-) retard (+)charged proteins:Cation exchange

Quaternary ammonium salts (+) retard (-)charged proteins:Anion exchange

Separations facilitated by adjusting pH


Affinity chromatographyAffinity chromatography

Stationary phase contains a species that has specific favorable interaction with the protein we want

DNA-binding protein specific to AGCATGCT: bind AGCATGCT to a column, and the protein we want will stick; every other protein falls through

Often used to purify antibodies by binding the antigen to the column


Metal-ion affinity Metal-ion affinity chromatographychromatography

Immobilize a metal ion, e.g. Ni, to the column material

Proteins with affinity to that metal will stick

Wash them off afterward with a ligand with an even higher affinity

We can engineer proteins to contain the affinity tag:poly-histidine at N- or C-terminus


High-performance liquid High-performance liquid chromatographychromatography

Many LC separations can happen faster and more effectively under high pressure

Works for small moleculesProtein application is routine too, both

for analysis and purificationFPLC is a trademark, but it’s used

generically


ElectrophoresisElectrophoresis

Separating analytes by charge by subjecting a mixture to a strong electric field

Gel electrophoresis: field applied to a semisolid matrix

Can be used for charge (directly) or size (indirectly)


SDS-PAGESDS-PAGE

Sodium dodecyl sulfate: strong detergent, applied to protein

Charged species binds quantitatively Denatures protein

– Good because initial shape irrelevant– Bad because it’s no longer folded

Larger proteins move slower because they get tangled in the matrix

1/Velocity √MW


SDS PAGE illustratedSDS PAGE illustrated


Isoelectric focusing IIsoelectric focusing I

Protein applied to gel without charged denaturant

Electric field set up over a pH gradient (typically pH 2 to 12)

Protein will travel until it reaches the pH where charge =0 (isoelectric point)


Isoelectric focusing IIIsoelectric focusing II

Sensitive to single changes in charge (e.g. asp -> asn)

Can be readily used preparatively with samples that are already semi-pure


Ultraviolet spectroscopyUltraviolet spectroscopy

Tyr, trp absorb and fluoresce:abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr)

Reliable enough to use for estimating protein concentration via Beer’s law

UV absorption peaks for cofactors in various states are well-understood

More relevant for identification of moieties than for structure determination

Quenching of fluorescence sometimes provides structural information


Warning: Specialty Content!Warning: Specialty Content!

I determine protein structures (and develop methods for determining protein structures) as my own research focus

So it’s hard for me to avoid putting a lot of emphasis on this material

But today I’m allowed to do that, because it’s one of the stated topics of the day.


How do we determine structure?How do we determine structure?

We can distinguish between methods that require little prior knowledge (crystallography, NMR, ?CryoEM?)and methods that answer specific questions (XAFS, fiber, …)

This distinction isn’t entirely clear-cut


Crystallography: overviewCrystallography: overview

Crystals are translationally ordered 3-D arrays of molecules

Conventional solids are usually crystalsProteins have to be coerced into

crystallizing… but once they’re crystals, they

behave like other crystals, mostly


How are protein crystals How are protein crystals unusual?unusual?

Aqueous interactions required for crystal integrity: they disintegrate if dried

Bigger unit cells (~10nm, not 1nm)Small # of unit cells and static disorder

means they don’t scatter terribly wellSo using them to determine 3D

structures is feasible but difficult


Crystal structures: Fourier Crystal structures: Fourier transforms of diffraction resultstransforms of diffraction results Experiment:

– Grow crystal, expose it to X-ray– Record diffraction spots– Rotate through small angle and repeat ~180 times

Position of spots tells you size, shape of unit cell

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


What are these What are these FFhklhkl values? values?

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)

Critical point: any single diffraction spot contains information derived from all the atoms in the structure; and any atom contributes to all the diffraction spots


The phase problemThe phase problem

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

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

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!

Fhkl

ahkl

bhkl


What can we learn?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 non-H atoms, most

solvent, some disordered atoms; some H’s


What does this look like?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


How’s the field changing?How’s the field changing?

1990: all structures done by professionalsNow: 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– Most structures will be determined by cell

biologists & molecular biologists


Macromolecular NMRMacromolecular NMR

NMR is a mature field Depends on resonant interaction between EM

fields and unpaired nucleons (1H, 15N, 31S) Raw data yield interatomic distances Conventional spectra of proteins are too

muddy to interpret Multi-dimensional (2-4D) techniques:

initial resonances coupled with additional ones


Typical protein 2-D spectrumTypical protein 2-D spectrum

Challenge: identify whichH-H distance is responsible for a particular peak

Enormous amount of hypothesis testing required

Prof. Mark Searle,University of Nottingham


Results of NMR studiesResults of NMR studies

Often there’s a family of structures that satisfy the NMR data equally well

Can be portrayed as a series of threads tied down at unambiguous assignments

They portray the protein’s structure in solution

The ambiguities partly represent real molecular diversity; but they also represent atoms that area in truth well-defined, but the NMR data don’t provide the unambiguous assignment


Comparing NMR to X-rayComparing NMR to X-ray

NMR family of structures often reflects real conformational heterogeneity

Nonetheless, it’s hard to visualize what’s happening at the active site at any instant

Hydrogens sometimes well-located in NMR;they’re often the least defined atoms in an X-ray structure

The NMR structure is obtained in solution! Hard to make NMR work if MW > 35 kDa


What does it mean when NMR What does it mean when NMR and X-ray structures differ?and X-ray structures differ?

Lattice forces may have tied down or moved surface amino acids in X-ray structure

NMR may have errors in it X-ray may have errors in it (measurable) X-ray structure often closer to true atomic

resolution X-ray structure has built-in reliability checks


Cryoelectron Cryoelectron microscopymicroscopy

Like X-ray crystallography,EM damages the samples

Samples analyzed < 100Ksurvive better

2-D arrays of molecules– Spatial averaging to improve

resolution– Discerning details ~ 4Å resolution

Can be used with crystallography


Circular dichroismCircular dichroism Proteins in solution can

rotate polarized light Amount of rotation varies

with Effect depends on

interaction with secondary structure elements, esp.

Presence of characteristic patterns in presence of other stuff enables estimate of helical content

protein methods andy howard introductory biochemistry fall 2010, iit

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