Protein Methods & Functions

1. Protein Methods & Functions Andy HowardBiochemistry Lectures, Spring 2019Tuesday 5 February 2019

2. How to study proteins; what they do • We will complete our discussion of protein methods today • Not all proteins are enzymes, but many are; here we’ll discuss the numerous roles that proteins play in biology Protein Methods & Functions

3. Methods Purification Characterization Structure Moonlighting Protein functions Structure Enzymes Electron-transport Storage & transport Hormones Receptors Topics for today Protein Methods & Functions

4. Electrophoresis (CF&M§5.3) • 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) Protein Methods & Functions

5. SDS-PAGE • Sodium dodecyl sulfate: strong detergent, applied to protein • Charged species binds quantitatively • Denatures protein • Good: initial shape irrelevant • Bad: it’s no longer folded Protein Methods & Functions

6. How SDS can tell us mol. weight Cf. CF&M fig. 5.12 Log10(mol wt) ElectrophoreticVelocity Larger proteins move slower because they get tangled in the matrix:log(MW) = -mx + b, wherex = electrophoretic velocity Protein Methods & Functions

7. SDS PAGE illustrated Cf. CF&M fig. 5.13 Protein Methods & Functions

8. Isoelectric focusing • 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 wherecharge =0 (isoelectric point) Protein Methods & Functions

9. Using Isoelectric Focusing Sensitive to single changes in charge (e.g. asp asn) Readily used preparatively with samples that are already semi-pure Protein Methods & Functions

10. Applying this method Spectroscopy is more relevant for identification of moieties than for structure determination Quenching of fluorescence sometimes provides structural information Protein Methods & Functions

11. Mass spectrometry as an analytical tool • Mass spectrometry separates molecular species according to their mass/charge value • It’s been used in chemistry for a century but couldn’t be applied to proteins until two techniques where developed in the 1980’s that preserved their properties: • Electrospray and MALDI; Cf. CF&M section 5A Protein Methods & Functions

12. iClicker quiz question 1 1. A protein has pI = 4. At pH=7 its charge will be • (a) positive • (b) negative • (c) neutral • (d) insufficient information provided. Protein Methods & Functions

13. iClicker question 2 2. Which of the following techniques does not separate proteins by size? • (a) SDS-PAGE • (b) Size-exclusion • (c) Isoelectric focusing • (d) Mass spectrometry • (e) All four of these separate by size. Protein Methods & Functions

14. Structure Methods! . . .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. Protein Methods & Functions

15. How do we determine structure? (CF&M §4.4) • 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 Protein Methods & Functions

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 Protein Methods & Functions

17. How are protein crystals unusual? • Aqueous interactions required for crystal integrity: they disintegrate if dried • Bigger unit cells (~10nm, not 1nm) • Intermolecular forces are weak ionic forces • Small # of unit cells and static disorder means they don’t scatter terribly well • Determining 3D structures is feasible but difficult Protein Methods & Functions

18. Crystal 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(2ik•x) = cos2k•x + isin2k•x Protein Methods & Functions

19. Relating ρ(r) to intensities 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) hklFhkl exp(-2ih•r) Inverse of that:Fhkl = ⎰V(r) exp(2ih•r) Protein Methods & Functions

20. Fhkl bhkl  The phase problem ahkl • 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(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! Protein Methods & Functions

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 • Realistic resolution usually poorer than that Protein Methods & Functions

22. What else can we learn? Hydrogen positions can be inferred, especially if you are able to get high-resolution data (see next slide) Atomic mobility can estimated for intermediate to high resolution data Protein Methods & Functions

23. Limitations of resolution • Low values of Dmin mean more detail and better understanding • Often the crystal doesn’t diffract ideally, so Dmin is larger than l/2—1.5Å, 2.5Å, or 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 Protein Methods & Functions

24. 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 Protein Methods & Functions

25. 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 others Protein Methods & Functions

26. Typical protein 2-D spectrum • Challenge: identify which H-H distance is responsible for a particular peak • Enormous amount of hypothesis testing required Prof. Mark Searle,University of Nottingham Protein Methods & Functions

27. Results • 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 • 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 Protein Methods & Functions

28. 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 Protein Methods & Functions

29. NMR vs. X-ray, continued The NMR structure is obtained in solution! Hard to make NMR work if MW > 55 kDa, and even when you can, it takes a lot of computer time Protein Methods & Functions

30. 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 Protein Methods & Functions

31. 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 Protein Methods & Functions

32. Solution scattering • Proteins in solution scatter X-raysin characteristic ways • Low-resolution structural information available • Does not require crystals • Until ~ 2000 you needed high [protein] • Thanks to BioCAT, SAXS on dilute proteins is becoming more feasible • Hypothesis-based analysis Protein Methods & Functions

33. Fiber Diffraction • Some proteins, like many DNA molecules, possessapproximate fibrous order(2-D ordering) • Produce characteristic fiber diffraction patterns • Collagen, muscle proteins, filamentous viruses Protein Methods & Functions

34. X-ray spectroscopy • All atoms absorb UV or X-rays at characteristic wavelengths • Higher Z means higher energy,lower for a particular edge • Perturbation of absorption spectra at E = Epeak +  yields neighbor info • Changes just below the peak yield oxidation-state information • X-ray relevant for metals, Se, I Protein Methods & Functions

35. Mass spectrometry as a structural tool • MS tells you molecular weights • Can give high precision in m/m • Not inherently a way of determining structure • Can distinguish oligomeric state • Coupled with proteolytic digestion, it can be used to find fragmentation patterns Protein Methods & Functions

36. Circular dichroism • Proteins in solution can rotate polarized light • Amount of rotation varies with  • Effect depends on interaction with secondary structure elements, esp.  Protein Methods & Functions

37. How to use CD for structure Presence of characteristic  patterns in presence of other stuff enables estimate of helical content Protein Methods & Functions

38. iClicker question 3 5W8J 1T2F 3. Which of these structures provides a more detailed and precise knowledge of the structure of lactate dehydrogenase? • (a) PDB 1T2F, Dmin=3Å • (b) PDB 5W8J, Dmin=1.55Å • (c) both are equally informative • (d) insufficient information to tell Protein Methods & Functions

39. iClicker question 4 4. Shown are structures of myoglobin and an Fab component of an immunoglobulin. Which of these could be most effectively studied via circular dichroism? • Myoglobin • Fab component • CD will work for both • CD is useless for both Myoglobin, PDB 5YCE 4-4-20 Fab, PDB 1FLR Protein Methods & Functions

40. Protein function • We’ll devote most of the rest of the lecture to a discussion of the various functions performed by proteins • We’ll do a quick run-through of the various functions, and then discuss PTM and allostery. Protein Methods & Functions

41. Classes of proteins • Next segment of this lecture:small encyclopedia of theprotein functions • Reminder:proteins can take onmore than one function Arginosuccinate lyase / Delta crystallinPDB 1auw, 2.5Å206kDa tetramer Protein Methods & Functions

42. Moonlighting proteins A protein may evolve for one purpose … then it gets co-opted for another Studies by C. Jeffery et al Protein Methods & Functions

43. Structural proteins • Mechanical or scaffolding tasks • Don’t do chemistry, unless this is a chemical reaction:(Person standing upright) (Person lying in a puddle on the floor) • Examples: collagen, fibroin, keratin • Often enzymes are recruited to perform structural roles CollagenmodelPDB 1K6F Protein Methods & Functions

44. Enzymes • Enzymes are biological catalysts, i.e. their job is to reduce the activation energy barrier between substrates and products • Tend to be at least 12kDa (why?You need that much scaffolding) • Usually but not always aqueous • Usually organized with hydrophilic residues facing outward hen egg-white lysozyme 14.2kDa monomer PDB 2vb1, 0.65Å Protein Methods & Functions

45. Electron-transport proteins • Involved in oxidation-reductionreactions via • Incorporated metal ions • Small organic moieties (NAD, FAD) • Generally not enzymes: they’re ultimately altered by their reactions Recombinant human cytochrome cPDB 1J3SNMR structure11.4kDa Protein Methods & Functions

46. But they can be parts of larger enzyme systems! Some participate in larger enzyme complexes than can restore them to their original state Protein Methods & Functions

47. Sizes and characteristics • Some ET proteins: fairly small • Cytochrome c • Some flavodoxins • Others are multi-polypeptide complexes Anacystisflavodoxin PDB 1czn1.7Å18.6 kDa Protein Methods & Functions

48. Coenzymes and metals in electron-transport proteins Coenzymes or metal ions may be closely associated (covalent in cytochromes) or more loosely bound Protein Methods & Functions

49. Storage and transport proteins • Hemoglobin, myoglobin classic examples • “honorary enzymes”: share some characteristics with enzymes • Sizes vary widely Sperm-whale myoglobin18kDa monomerPDB 1MTJ, 1.7Å Protein Methods & Functions

50. Intracellular transporters Many transporters operate over much smaller size-scales than hemoglobin(µm vs. m): often involved in transport across membranes We’ll discuss intracellular transport a lot! Protein Methods & Functions