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Protein Structure & Function Andy Howard Introductory Biochemistry, Fall 2010 7 September 2010 Biochem: Protein Functions I 09/07/2010 Proteins and enzymes Proteins perform a variety of functions, including acting as enzymes. 09/07/2010 Biochem: Protein Functions I p. 2 of 52 Plans for Today Secondary Structure Types Helices Sheets Disulfides Tertiary Structures Quaternary Structure Visualizing structure 09/07/2010 The Protein Data Bank Tertiary & quaternary structure Protein Functions Structure-function relationships Post-translational modification Biochem: Protein Functions I p. 3 of 52 Components of secondary structure , 310, helices pleated sheets and the strands that comprise them Beta turns More specialized structures like collagen helices 09/07/2010 Biochem: Protein Functions I p. 4 of 52 An accounting for secondary structure: phospholipase A2 09/07/2010 Biochem: Protein Functions I p. 5 of 52 Alpha helix 09/07/2010 Biochem: Protein Functions I p. 6 of 52 Characteristics of helices 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 09/07/2010 Biochem: Protein Functions I p. 7 of 52 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 09/07/2010 Biochem: Protein Functions I p. 8 of 52 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 Nterminal end of an otherwise -helix helix: even rarer: NH to C=O five residues earlier 09/07/2010 Biochem: Protein Functions I p. 9 of 52 Beta strands Structures containing roughly extended polypeptide strands Extended conformation stabilized by interstrand main-chain hydrogen bonds No defined interval in sequence number between amino acids involved in H-bond 09/07/2010 Biochem: Protein Functions I p. 10 of 52 Sheets: roughly planar Folds straighten H-bonds Side-chains roughly perpendicular from sheet plane Consecutive side chains up, then down Minimizes intra-chain collisions between bulky side chains 09/07/2010 Biochem: Protein Functions I p. 11 of 52 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 09/07/2010 Biochem: Protein Functions I p. 12 of 52 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 09/07/2010 Biochem: Protein Functions I p. 13 of 52 Beta turns Abrupt change in direction , angles are characteristic of beta Main-chain H-bonds maintained almost all the way through the turn Jane Richardson and others have characterized several types 09/07/2010 Biochem: Protein Functions I p. 14 of 52 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 09/07/2010 Biochem: Protein Functions I p. 15 of 52 Note about disulfides H Cysteine residues brought S into proximity under H C oxidizing conditions can form a disulfide Forms a “cystine” residue Oxygen isn’t always the oxidizing agent Can bring sequence-distant residues close together and stabilize the protein 09/07/2010 Biochem: Protein Functions I H S H H + (1/2)O 2 H2O H H C C S H S H p. 16 of 52 H C 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 ~ 1- 4 kcal/mol 09/07/2010 Biochem: Protein Functions I p. 17 of 52 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 09/07/2010 Biochem: Protein Functions I p. 18 of 52 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 09/07/2010 Biochem: Protein Functions I p. 19 of 52 Silk fibroin Antiparallel beta sheets running parallel to the silk fiber axis Multiple repeats of (Gly-Ser-GlyAla-Gly-Ala)n 09/07/2010 Biochem: Protein Functions I p. 20 of 52 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” 09/07/2010 Biochem: Protein Functions I p. 21 of 52 Tertiary Structure 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, . . . 09/07/2010 Biochem: Protein Functions I p. 22 of 52 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 09/07/2010 Biochem: Protein Functions I p. 23 of 52 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! 09/07/2010 Biochem: Protein Functions I p. 24 of 52 Protein Topology Description of the connectivity of segments of secondary structure and how they do or don’t cross over 09/07/2010 Biochem: Protein Functions I p. 25 of 52 TIM barrel Alternating , creates parallel -pleated sheet Bends around as it goes to create barrel 09/07/2010 Biochem: Protein Functions I p. 26 of 52 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 09/07/2010 Biochem: Protein Functions I p. 27 of 52 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 09/07/2010 Biochem: Protein Functions I p. 28 of 52 How do we show 3-D? Stereo pairs Rely on the way the brain processes leftand right-eye images If we allow our eyes to go slightly walleyed or crossed, the image appears three-dimensional Dynamics: rotation of flat image Perspective (hooray, Renaissance) 09/07/2010 Biochem: Protein Functions I p. 29 of 52 Straightforward example Sso7d bound to DNA Gao et al (1998) NSB 5: 782 09/07/2010 Biochem: Protein Functions I p. 30 of 52 A little more complex: Aligning Cytochrome C5 with Cytochrome C550 09/07/2010 Biochem: Protein Functions I p. 31 of 52 Stereo pair: Release factor 2/3 Klaholz et al, Nature (2004) 427:862 09/07/2010 Biochem: Protein Functions I p. 32 of 52 Ribbon diagrams Mostly helical: E.coli RecG - DNA PDB 1gm5 3.24Å, 105 kDa QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 09/07/2010 Mixed: hen egg-white lysozyme PDB 2vb1 0.65Å, 14.2kDa QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Biochem: Protein Functions I p. 33 of 52 The Protein Data Bank http://www.rcsb.org/ This is an electronic repository for threedimensional structural information of polypeptides and polynucleotides 67656 structures as of September 2010 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-alpha-helical proteins 09/07/2010 Biochem: Protein Functions I p. 34 of 52 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 09/07/2010 Biochem: Protein Functions I p. 35 of 52 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 09/07/2010 Biochem: Protein Functions I p. 36 of 52 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 09/07/2010 Biochem: Protein Functions I p. 37 of 52 Domains Proteins (including singlepolypeptide proteins) often contain roughly selfcontained domains Domains often separated by linkers Linkers sometimes flexible or extended or both Cf. fig. 6.36 in G&G 09/07/2010 Biochem: Protein Functions I p. 38 of 52 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 09/07/2010 Biochem: Protein Functions I p. 39 of 52 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! 09/07/2010 Biochem: Protein Functions I p. 40 of 52 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. 09/07/2010 Biochem: Protein Functions I p. 41 of 52 Structure-function relationships Proteins with known function: structure can tell is how it does its job Example: yeast alcohol dehydrogenase: Catalyzes ethanol + 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 09/07/2010 Biochem: Protein Functions I p. 42 of 52 Why this example? Structures of ADH from QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. several eukaryotic and prokaryotic organisms already known Yeast ADH Yeast ADH is clearly PDB 2hcy important and heavily 2.44Å 152 kDa tetramer studied, but until 2006: no dimer shown structure! We got crystals 11 years ago, but so far I haven’t been able 09/07/2010 p. 43 of 52 Biochem: Functions I to determine theProtein structure 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) 09/07/2010 Biochem: Protein Functions I p. 44 of 52 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 09/07/2010 Biochem: Protein Functions I p. 45 of 52 Structure-function relationships II Protein with unknown function: structure might tell us what the function is! Generally we accomplish this by recognizing structural similarity to another protein whose function is known Sometimes we get lucky: we can figure it out by binding of a characteristic cofactor 09/07/2010 Biochem: Protein Functions I p. 46 of 52 What proteins can do: I 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. 09/07/2010 Biochem: Protein Functions I p. 47 of 52 What proteins can do: II 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 09/07/2010 Biochem: Protein Functions I p. 48 of 52 What proteins can do: III Proteins can readily bind organic, metallic, or organometallic ligands called cofactors. These extend the functionality of proteins well beyond the chemical nimbleness that polypeptides by themselves can accomplish We’ll study these cofactors in detail in chapter 17 09/07/2010 Biochem: Protein Functions I p. 49 of 52 Zymogens and PTM Many proteins are synthesized on the ribosome in an inactive form, viz. as a zymogen The conversions that alter the ribosomally encoded protein into its active form is an instance of post-translational modification 09/07/2010 Biochem: Protein Functions I QuickTime™ and a decompressor are needed to see this picture. PDB 3CNQ Subtilisin prosegment complexed with subtilisin 1.71Å; 35 kDa monomer p. 50 of 52 Why PTM? This happens for several reasons Active protein needs to bind cofactors, ions, carbohydrates, and other species Active protein might be dangerous at the ribosome, so it’s created in inactive form and activated elsewhere Proteases (proteins that hydrolyze peptide bonds) are examples of this phenomenon … but there are others 09/07/2010 Biochem: Protein Functions I p. 51 of 52 Protein Phosphorylation Most common form of PTM that affects just one amino acid at a time Generally involves phosphorylating side chains of specific polar amino acids: mostly S,T,Y,H (and D, E) Enzymes that phosphorylate proteins are protein kinases and are ATP or GTP dependent Enzymes that remove phosphates are phosphatases and are ATP and GTP independent 09/07/2010 Biochem: Protein Functions I p. 52 of 52