Protein Methods II

1. Protein Methods II Andy Howard Introductory BiochemistryFall 2009, IIT

2. Proteins are worth studying • We’ll finish our quick overview of methods of studying proteins Biochemistry: Protein Methods II

3. Plans • Purification methods • Analytical methods • Structural methods Biochemistry: Protein Methods II

4. Ion-exchange chromatography • Charged species affixed to column • Phosphonates (-) retard (+)charged proteins:Cation exchange • Quaternary ammonium salts (+) retard (-)charged proteins: Anion exchange • Separations facilitated by adjusting pH Biochemistry: Protein Methods II

5. Affinity 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 Biochemistry: Protein Methods II

6. Metal-ion affinity chromatography • 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 Biochemistry: Protein Methods II

7. High-performance liquid chromatography • Many LC separations can happen faster and more effectively under high pressure • Works for small molecules • Protein application is routine too, both for analysis and purification • FPLC is a trademark, but it’s used generically Biochemistry: Protein Methods II

8. Electrophoresis • 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) Biochemistry: Protein Methods II

9. SDS-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 Biochemistry: Protein Methods II

10. SDS PAGE illustrated Biochemistry: Protein Methods II

11. Isoelectric 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) Biochemistry: Protein Methods II

12. Isoelectric focusing II • Sensitive to single changes in charge (e.g. asp -> asn) • Can be readily used preparatively with samples that are already semi-pure Biochemistry: Protein Methods II

13. Ultraviolet 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 Biochemistry: Protein Methods II

14. 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. Biochemistry: Protein Methods II

15. 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 Biochemistry: Protein Methods II

16. Crystallography: overview • Crystals are translationally ordered 3-D arrays of molecules • Conventional solids are usually crystals • Proteins have to be coerced into crystallizing • … but once they’re crystals, they behave like other crystals, mostly Biochemistry: Protein Methods II

17. How are protein crystals 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 well • So using them to determine 3D structures is feasible but difficult Biochemistry: Protein Methods II

18. Crystal structures: Fourier transforms 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(2ik•x) • Therefore intensity Ihkl = C*|Fhkl|2 Biochemistry: Protein Methods II

19. What are these Fhkl values? • Fhkl is a complex coefficient in the Fourier transform of the electron density in the unit cell:(r) = (1/V) hklFhkl exp(-2ih•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 Biochemistry: Protein Methods II

20. The phase problem Fhkl • Note that we saidIhkl = 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(ihkl) • 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! bhkl  ahkl Biochemistry: Protein Methods II

21. 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 Biochemistry: Protein Methods II

22. 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 Biochemistry: Protein Methods II

23. 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 • Most structures will be determined by cell biologists & molecular biologists Biochemistry: Protein Methods II

24. Macromolecular 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 Biochemistry: Protein Methods II

25. Typical 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 Biochemistry: Protein Methods II

26. Results 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 Biochemistry: Protein Methods II

27. Comparing 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 Biochemistry: Protein Methods II

28. What does it mean when NMR 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 Biochemistry: Protein Methods II

29. Cryoelectron microscopy • 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 Biochemistry: Protein Methods II

30. Circular 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 Biochemistry: Protein Methods II

31. Poll question: discuss! Sperm whale myoglobinPDB 2jho1.4Å16.9 kDa • 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 Anti-fluorescein FabPDB 1flr1.85 Å52 KDa Biochemistry: Protein Methods II

32. Ultraviolet 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 Biochemistry: Protein Methods II

33. X-ray spectroscopy • All atoms absorb UV orX-rays at characteristic wavelengths • Higher Z means higher energy, lower for a particular edge Biochemistry: Protein Methods II

34. X-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 Biochemistry: Protein Methods II

35. Solution 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 Biochemistry: Protein Methods II

36. Fiber Diffraction • Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering) • Produce characteristic fiber diffraction patterns • Collagen, muscle proteins, filamentous viruses Biochemistry: Protein Methods II