Protein Structures and Methods

1. Protein Structures and Methods Andy Howard Introductory Biochemistry15 September 2014, IIT

2. Proteins are worth studying • We’ll offer an overview of what proteins look like • Then we’ll perform a quick overview of methods of studying proteins • Purification methods • Analytical methods • Structural methods Protein Fundamentals

3. Levels of protein structure Primary Secondary Tertiary Quaternary Domains TIM Barrels Generalizations about structure Methods of purifying proteins Plans Protein Fundamentals

4. Levels of Protein Structure:G&G §5.1 • We conventionally describe proteins at four levels of structure, from most local to most global: • Primary: linear sequence of peptide units and covalent disulfide bonds • Secondary: main-chain H-bonds that define short-range order in structure • Tertiary: three-dimensional fold of a polypeptide • Quaternary: Folds of multiple polypeptide chains to form a complete oligomeric unit Protein Fundamentals

5. Components of secondary structure (G&G §6.3) • , 310,  helices • pleated sheets and the strands that comprise them • Beta turns • More specialized structures like collagen helices Protein Fundamentals

6. An accounting for secondary structure: phospholipase A2 Protein Fundamentals

7. Alpha helix (G&G fig. 6.6) Protein Fundamentals

8. Characteristics of  helices(G&G Fig. 6.9) • Hydrogen bonding from amino nitrogen to carbonyl oxygen in the residue 4 earlier in the chain • 3.6 residues per turn • Amino acid side chains face outward, for the most part • ~ 10 residues long in globular proteins Protein Fundamentals

9. What would disrupt this? • Not much: the side chains don’t bump into one another • Proline residue will disrupt it: • Main-chain N can’t H-bond • The ring forces a kink • Glycines sometimes disrupt because they tend to be flexible Protein Fundamentals

10. Other helices • NH to C=O four residues earlier is not the only pattern found in proteins • 310 helix is NH to C=O three residues earlier • More kinked; 3 residues per turn • Often one H-bond of this kind at N-terminal end of an otherwise -helix •  helix: even rarer: NH to C=O five residues earlier Protein Fundamentals

11. Beta strands • Structures containing roughly extended polypeptide strands • Extended conformation stabilized by inter-strand main-chain hydrogen bonds • No defined interval in sequence number between amino acids involved in H-bond Protein Fundamentals

12. Sheets: roughly planar(G&G fig. 6.10) • Pleats straighten H-bonds • Side-chains roughly perpendicular from sheet plane • Consecutive side chains up, then down • Minimizes intra-chain collisions between bulky side chains Protein Fundamentals

13. Anti-parallel beta sheet • Neighboring strands extend in opposite directions • Complementary C=O…N bonds from top to bottom and bottom to top strand • Slightly pleated for optimal H-bond strength Protein Fundamentals

14. Parallel Beta Sheet • N-to-C directions are the same for both strands • You need to get from the C-end of one strand to the N-end of the other strand somehow • H-bonds at more of an angle relative to the approximate strand directions • Therefore: more pleated than anti-parallel sheet Protein Fundamentals

15. Beta turns • Abrupt change in direction • , angles arecharacteristic of beta • Main-chain H-bonds maintained almost all the way through the turn • Jane Richardson and others have characterized several types Protein Fundamentals

16. Collagen triple helix • Three left-handed helical strands interwoven with a specific hydrogen-bonding interaction • Every 3rd residue approaches other strands closely: so they’re glycines Protein Fundamentals

17. Hydrogen bonds, revisited • Protein settings, H-bonds are almost always: • Between carbonyl oxygen and hydroxyl:(C=O ••• H-O-) • between carbonyl oxygen and amine:(C=O ••• H-N-) • –OH to –OH, –OH to –NH, … less significant • These are stabilizing structures • Any stabilization is (on its own) entropically disfavored; • Sufficient enthalpic optimization overcomes that! • In general the optimization is ~ 3-7 kJ/mol Protein Fundamentals

18. Secondary structures in structural proteins • Structural proteins often have uniform secondary structures • Seeing instances of secondary structure provides a path toward understanding them in globular proteins • Examples: • Alpha-keratin (hair, wool, nails, …):-helical • Silk fibroin (guess) is -sheet Protein Fundamentals

19. Alpha-keratin • Actual -keratins sometimes contain helical globular domains surrounding a fibrous domain • Fibrous domain: long segments of regular -helical bonding patterns • Side chains stick out from the axis of the helix Protein Fundamentals

20. Silk fibroin • Antiparallel beta sheets running parallel to the silk fiber axis • Multiple repeats of (Gly-Ser-Gly-Ala-Gly-Ala)n Protein Fundamentals

21. Secondary structure in globular proteins • Segments with secondary structure are usually short: 2-30 residues • Some globular proteins are almost all helical, but even then there are bends between short helices • Other proteins: mostly beta • Others: regular alternation of ,  • Still others: irregular , , “coil” Protein Fundamentals

22. Tertiary Structure(G&G §6.4) • The overall 3-D arrangement of atoms in a single polypeptide chain • Made up of secondary-structure elements & locally unstructured strands • Described in terms of sequence, topology, overall fold, domains • Stabilized by van der Waals interactions, hydrogen bonds, disulfides, . . . Protein Fundamentals

23. 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 Protein Fundamentals

24. Hydrophobic in, -philic out • Aqueous proteins arrange themselves so that polar groups are solvent-accessible and apolar groups are not • The energetics of protein folding are strongly driven by this hydrophobic in, hydrophilic out effect • Exceptions are membrane proteins Protein Fundamentals

25. Quaternary structure • Arrangement of individual polypeptide chains to form a complete oligomeric, functional protein • Individual chains can be identical or different • If they’re the same, they can be coded for by the same gene • If they’re different, you need more than one gene Protein Fundamentals

26. 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:often the monomer can’t do it on its own Protein Fundamentals

27. Not all proteins have all four levels of structure • Monomeric proteins don’t have quaternary structure • Tertiary structure: subsumed into 2ndry structure for many structural proteins (keratin, silk fibroin, …) • Some proteins (usually small ones) have no definite secondary or tertiary structure; they flop around! Protein Fundamentals

28. Protein Topology • Description of the connectivity of segments of secondary structure and how they do or don’t cross over Protein Fundamentals

29. TIM barrel • Alternating ,  creates parallel -pleated sheet • Bends around as it goes to create barrel Protein Fundamentals

30. 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 Protein Fundamentals

31. 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 Protein Fundamentals

32. 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) Protein Fundamentals

33. Green fluorescent protein Yang et al. (1999) Nature Biotechnology 14:1246 Protein Fundamentals

34. A little more complex Endonuclease V (Dalhus et al (2009) NSMB 16:138) Protein Fundamentals

35. Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862 Protein Fundamentals

36. A little more complex: AligningCytochrome C5 & Cytochrome C550 Protein Fundamentals

37. Mixed:hen egg-white lysozyme PDB 2vb10.65Å, 14.2kDa Mostly helical:E.coli RecG – DNAPDB 1gm53.24Å, 105 kDa Ribbon diagrams Protein Fundamentals

38. The Protein Data Bank • http://www.rcsb.org/ • This is an electronic repository for three-dimensional structural information of polypeptides and polynucleotides • 103015 structures as of September 2013 • 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-α-proteins Protein Fundamentals

39. What 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 Protein Fundamentals

40. 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. 6.38 in G&G Protein Fundamentals

41. Protein 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 Protein Fundamentals

42. Salting 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. Protein solubility, mg/ML 0 2 [Salt], M Protein Fundamentals

43. How 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 Protein Fundamentals

44. Dialysis • 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. Protein Fundamentals

45. Gel-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 Protein Fundamentals

46. 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 Protein Fundamentals

47. 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 Protein Fundamentals