protein methods
DESCRIPTION
Protein Methods. Andy Howard Introductory Biochemistry Fall 2010, IIT. Proteins are worth studying. We’ll perform a quick overview of methods of studying proteins Purification methods Analytical methods Structural methods. The Protein Data Bank. http://www.rcsb.org/ - PowerPoint PPT PresentationTRANSCRIPT
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
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The Protein Data BankThe Protein Data Bankhttp://www.rcsb.org/This is an electronic repository
for three-dimensional structural information of polypeptides and polynucleotides
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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
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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
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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
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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.
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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
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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.
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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
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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
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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
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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
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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
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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)
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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
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SDS PAGE illustratedSDS PAGE illustrated
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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)
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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