Protein Structure & Function

Protein Structure & Function Andy HowardIntroductory Biochemistry, Fall 2010 7 September 2010 Biochem: Protein Functions I

Proteins and enzymes • Proteins perform a variety of functions, including acting as enzymes. Biochem: Protein Functions I

Secondary Structure Types Helices Sheets Disulfides Tertiary Structures Quaternary Structure Visualizing structure The Protein Data Bank Tertiary & quaternary structure Protein Functions Structure-function relationships Post-translational modification Plans for Today Biochem: Protein Functions I

Components of secondary structure • , 310,  helices • pleated sheets and the strands that comprise them • Beta turns • More specialized structures like collagen helices Biochem: Protein Functions I

An accounting for secondary structure: phospholipase A2 Biochem: Protein Functions I

Alpha helix Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

Note about disulfides • Cysteine residues brought into proximity under 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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

Silk fibroin • Antiparallel beta sheets running parallel to the silk fiber axis • Multiple repeats of (Gly-Ser-Gly-Ala-Gly-Ala)n Biochem: Protein Functions I

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” Biochem: Protein Functions I

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, . . . Biochem: Protein Functions I

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 Biochem: Protein Functions I

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! Biochem: Protein Functions I

Protein Topology • Description of the connectivity of segments of secondary structure and how they do or don’t cross over Biochem: Protein Functions I

TIM barrel • Alternating ,  creates parallel -pleated sheet • Bends around as it goes to create barrel Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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) Biochem: Protein Functions I

Straightforward example • Sso7d bound to DNAGao et al (1998) NSB 5: 782 Biochem: Protein Functions I

A little more complex: • Aligning Cytochrome C5with Cytochrome C550 Biochem: Protein Functions I

Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862 Biochem: Protein Functions I

Mostly helical:E.coli RecG - DNA PDB 1gm53.24Å, 105 kDa Mixed:hen egg-white lysozyme PDB 2vb10.65Å, 14.2kDa Ribbon diagrams Biochem: Protein Functions I

The Protein Data Bank • http://www.rcsb.org/ • This is an electronic repository for three-dimensional 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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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! Biochem: Protein Functions I

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. Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 11 years ago, but so far I haven’t been able to determine the structure Yeast ADH PDB 2hcy2.44Å 152 kDa tetramerdimer shown Biochem: Protein Functions I

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) Biochem: Protein Functions I

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 Biochem: Protein Functions I

Structure-functionrelationships 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 Biochem: Protein Functions I

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. Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 Biochem: Protein Functions I

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 PDB 3CNQSubtilisin prosegment complexed with subtilisin1.71Å; 35 kDa monomer Biochem: Protein Functions I

Protein Structure & Function