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Protein Methods II. Andy Howard Introductory Biochemistry Fall 2009, IIT. Proteins are worth studying. We’ll finish our quick overview of methods of studying proteins. Plans. Purification methods Analytical methods Structural methods. Ion-exchange chromatography. - PowerPoint PPT Presentation
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Protein Methods IIProtein Methods II
Andy HowardIntroductory Biochemistry
Fall 2009, IIT
09/10/09 Biochemistry: Protein Methods II p. 2 of 36
Proteins are worth studyingProteins are worth studying
We’ll finish our quick overview of methods of studying proteins
09/10/09 Biochemistry: Protein Methods II p. 3 of 36
PlansPlans
Purification methodsAnalytical methodsStructural methods
09/10/09 Biochemistry: Protein Methods II p. 4 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 5 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 6 of 36
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 even higher affinity
We can engineer proteins to contain the affinity tag:poly-histidine at N- or C-terminus
09/10/09 Biochemistry: Protein Methods II p. 7 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 8 of 36
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)
09/10/09 Biochemistry: Protein Methods II p. 9 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 10 of 36
SDS PAGE illustratedSDS PAGE illustrated
09/10/09 Biochemistry: Protein Methods II p. 11 of 36
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)
09/10/09 Biochemistry: Protein Methods II p. 12 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 13 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 14 of 36
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.
09/10/09 Biochemistry: Protein Methods II p. 15 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 16 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 17 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 18 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 19 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 20 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 21 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 22 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 23 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 24 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 25 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 26 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 27 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 28 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 29 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 30 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 31 of 36
Poll question: Poll question: discuss!discuss!
Which protein would yield a more interpretable CD spectrum?– (a) myoglobin– (b) Fab fragment of
immunoglobulin G– (c) both would be fully
interpretable– (d) CD wouldn’t tell us
anything about either protein
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Anti-fluorescein FabPDB 1flr1.85 Å52 KDa
Sperm whale myoglobinPDB 2jho1.4Å16.9 kDa
09/10/09 Biochemistry: Protein Methods II p. 32 of 36
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
09/10/09 Biochemistry: Protein Methods II p. 33 of 36
X-ray spectroscopyX-ray spectroscopy
All atoms absorb UV orX-rays at characteristic wavelengths
Higher Z means higher energy, lower for a particular edge
09/10/09 Biochemistry: Protein Methods II p. 34 of 36
X-ray spectroscopy IIX-ray spectroscopy II
Perturbation of absorption spectra at E = Epeak + yields neighbor information
Changes just below the peak yield oxidation-state information
X-ray relevant for metals, Se, I
09/10/09 Biochemistry: Protein Methods II p. 35 of 36
Solution scatteringSolution scattering
Proteins in solution scatter X-rays in characteristic, spherically-averaged ways
Low-resolution structural information available
Does not require crystals Until ~ 2000: needed high [protein] Thanks to BioCAT, SAXS on dilute
proteins is becoming more feasible Hypothesis-based analysis
09/10/09 Biochemistry: Protein Methods II p. 36 of 36
Fiber Fiber DiffractionDiffraction
Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering)
Produce characteristic fiber diffraction patterns
Collagen, muscle proteins, filamentous viruses