Structure Methods; Protein Function

1. Structure Methods; Protein Function Andy HowardIntroductory Biochemistry, Fall 20089 September 2008 Biochemistry: Structure Methods

2. Special Aspects of Tertiary & Quaternary Structure Structural methods Computation X-ray Crystallography NMR Spectroscopy Cryoelectron Microscopy Other experimental techniques Protein Functions Post-translational modification Specific functions Structural proteins Enzymes Electron transport Storage & transport Proteins Hormones & receptors Nucleic-acid binding proteins Distributions Topics for today Biochemistry: Structure Methods

3. Protein Topology • Description of the connectivity of segments of secondary structure and how they do or don’t cross over Biochemistry: Structure Methods

4. TIM barrel • Alternating ,  creates parallel -pleated sheet • Bends around as it goes to create barrel Biochemistry: Structure Methods

5. Domains • Proteins (including single-polypeptide proteins) often contain roughly self-contained domains • Domains often separated by linkers • Linkers sometimes flexible or extended or both • Cf. fig. 6.36 in G&G Biochemistry: Structure Methods

6. Generalizations about Tertiary Structure • Most globular proteins contain substantial quantities of secondary structure • The non-secondary segments are usually short; few knots or twists • Most proteins fold into low-energy structures—either the lowest or at least in a significant local minimum of energy • Generally the solvent-accessible surface area of a correctly folded protein is small Biochemistry: Structure Methods

7. Generalizations about quaternary structure • Considerable symmetry in many quaternary structure patterns (see G&G section 6.5) • Weak polar and solvent-exclusion forces add up to provide driving force for association • Many quaternary structures are necessary to function: monomer can’t do it on its own in a lot of cases Biochemistry: Structure Methods

8. How do we visualize protein structures? • It’s often as important to decide what to omit as it is to decide what to include • Any segment larger than about 10Å needs to be simplified if you want to understand it • What you omit depends on what you want to emphasize Biochemistry: Structure Methods

9. Styles of protein depiction • All atoms • All non-H atoms • Main-chain (backbone) only • One dot per residue (typically at C) • Ribbon diagrams: • Helical ribbon for helix • Flat ribbon for strand • Thin string for coil Biochemistry: Structure Methods

10. How do we show 3-D? • Stereo pairs • Rely on the way the brain processes left- and right-eye images • If we allow our eyes to go slightly wall-eyed or crossed, the image appears three-dimensional • Dynamics: rotation of flat image • Perspective (hooray, Renaissance) Biochemistry: Structure Methods

11. Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862 Biochemistry: Structure Methods

12. A more pedestrian application • Sso7d bound to DNAGao et al (1998) NSB 5: 782 Biochemistry: Structure Methods

13. A little more complex: • Aligning Cytochrome C5with Cytochrome C550 Biochemistry: Structure Methods

14. Mostly helical:E.coli RecG - DNA PDB 1gm53.24Å, 105 kDa Mixed:hen egg-white lysozyme PDB 2vb10.65Å, 14.2kDa Ribbon diagrams Biochemistry: Structure Methods

15. 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 the stated topic of the day. Biochemistry: Structure Methods

16. 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: Structure Methods

17. 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: Structure Methods

18. 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: Structure Methods

19. 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) • 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) Biochemistry: Structure Methods

20. The phase problem Fhkl bhkl  • Note that we saidIhkl = C*|Fhkl|2 • That means we can figure out|Fhkl| = (1/C)√Ihkl • But we can’t figure out the direction ofF: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! ahkl Biochemistry: Structure Methods

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: Structure Methods

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: Structure Methods

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: Structure Methods

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: Structure Methods

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: Structure Methods

26. 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 • 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: Structure Methods

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: Structure Methods

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: Structure Methods

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: Structure Methods

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: Structure Methods

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

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: Structure Methods

33. Protein Function: Generalities • Proteins do a lot of different things. Why? • Well, they’re coded for by the ribosomal factories • … But that just backs us up to the question of why the ribosomal mechanism codes for proteins and not something else! Biochemistry: Structure Methods

34. Proteins are chemically nimble • The chemistry of proteins is flexible • Protein side chains can participate in many interesting reactions • Even main-chain atoms can play roles in certain circumstances. • Wide range of hydrophobicity available (from highly water-hating to highly water-loving) within and around proteins gives them versatility that a more unambiguously hydrophilic species (like RNA) or a distinctly hydrophobic species (like a triglyceride) would not be able to acquire. Biochemistry: Structure Methods

35. What proteins can do • Proteins can act as catalysts, transporters, scaffolds, signals, or fuel in watery or greasy environments, and can move back and forth between hydrophilic and hydrophobic situations. • Furthermore, proteins can operate either in solution, where their locations are undefined within a cell, or anchored to a membrane. • Membrane binding keeps them in place. • Function may occur within membrane or in an aqueous medium adjacent to the membrane Biochemistry: Structure Methods

36. Structure-function relationships • Proteins with known function: structure can tell is how it does its job • Example: yeast alcohol dehydrogenase • Catalyzesethanol + NAD+ acetaldehyde + NADH + H+ • We can say something general about the protein and the reaction it catalyzes without knowing anything about its structure • But a structural understanding should help us elucidate its catalytic mechanism • Protein with unknown function: structure might tell us what the function is! Biochemistry: Structure Methods

37. Why this example? • Structures of ADH from several eukaryotic and prokaryotic organisms already known • Yeast ADH is clearly important and heavily studied, but until 2006: no structure! • We got crystals 7 years ago, but so far I haven’t been able to determine the structure Yeast ADH PDB 2hcy2.44Å 152 kDa tetramerdimer shown Biochemistry: Structure Methods

38. What we know about this enzyme • Cell contains an enzyme that interconverts ethanol and acetaldehyde, using NAD as the oxidizing agent (or NADH as the reducing agent) • We can call it alcohol dehydrogenase or acetaldehyde reductase; in this instance the former name is more common, but that’s fairly arbitrary (contrast with DHFR) Biochemistry: Structure Methods

39. Size and composition • Tetramer of identical polypeptides • Total molecular mass = 152 kDa • We can do arithmetic: the individual polypeptides have a molecular mass of 38 kDa (347 aa). • Human is a bit bigger: 374 aa per subunit • Each subunit has an NAD-binding Rossmann fold over part of its structure Biochemistry: Structure Methods