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Protein Function

Protein Function. Andy Howard Introductory Biochemistry, Fall 2008 11 September 2008. Zymogens and Post-translational modification Allostery Specific protein functions Structural proteins Enzymes Electron transport. Specific functions (continued) Storage & transport Proteins

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Protein Function

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  1. Protein Function Andy HowardIntroductory Biochemistry, Fall 200811 September 2008 Biochemistry: Protein Function

  2. Zymogens and Post-translational modification Allostery Specific protein functions Structural proteins Enzymes Electron transport Specific functions (continued) Storage & transport Proteins Hormones & receptors Nucleic-acid binding proteins Other functions Distributions Topics for today Biochemistry: Protein Function

  3. 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 Bacillus amylo-liquifaciensSubtilisin prosegment complexed with subtilisinPDB 1spb2.0Å29.2+8.6 kDa Biochemistry: Protein Function

  4. 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 Biochemistry: Protein Function

  5. iClicker question 1 Why are digestive proteases usually synthesized as inactive zymogens? • (a) Because they are produced in one organ and used elsewhere • (b) Because that allows the active form to be smaller than the ribosomally encoded form • (c) To allow for gene amplification and diversity • (d) So that the protease doesn’t digest itself prior to performing its intended digestive function • (e) None of the above Biochemistry: Protein Function

  6. iClicker question 2 Which amino acids can be readily phosphorylated by kinases? • (a) asp, phe, gly, leu • (b) ser, thr, tyr, his • (c) leu, ile, val, phe • (d) arg, lys, gln, asn • (e) none of the above. Biochemistry: Protein Function

  7. iClicker question 3 Why are kinase reactions ATP- (or GTP-) dependent, whereas phosphorylase reactions are not? • (a) To ensure stereospecific addition of phosphate to the target • (b) To prevent wasteful hydrolysis of product • (c) Adding phosphate is endergonic; taking phosphate off is exergonic • (d) None of the above. Biochemistry: Protein Function

  8. Allostery • Formal definition:alterations in protein function that occur when the structure changes upon binding of small molecules • In practice: often the allosteric effector is the same species as the substrate: they’re homotropic effectors • … but not always: allostery becomes an effective way of characterizing third-party (heterotropic) activators and inhibitors Biochemistry: Protein Function

  9. v0 What allostery means [S] • Non-enzymatic proteins can be allosteric:hemoglobin’s affinity for O2 is influenced by the binding of O2 to other subunits • In enzymes: non-Michaelis-Menten kinetics (often sigmoidal) when the allosteric activator is also the substrate Biochemistry: Protein Function

  10. R and T states • Protein with multiple substrate binding sites is in T (“tense”) state in absence of ligand or substrate • Binding of ligand or substrate moves enzyme into R (“relaxed”) state where its affinity for substrate at other sites is higher • Binding affinity or enzymatic velocity can then rise rapidly as function of [S] • Once all the protein is converted to R state, ordinary hyperbolic kinetics take over Biochemistry: Protein Function

  11. Other effectors can influence RT transitions • Post-translational covalent modifiers often influence RT equilibrium • Phosphorylation can stabilize either the R or T state • Binding of downstream products can inhibit TR transition • Binding of alternative metabolites can stabilize R state Biochemistry: Protein Function

  12. Why does that make sense? • Suppose reactions are: (E)A  B  C  D • Binding D to enzyme E (the enzyme that converts A to B) will destabilize its R state, limiting conversion of A to B and (ultimately) reducing / stabilizing [D]: homeostasis! Biochemistry: Protein Function

  13. Alternative pathways • Often one metabolite has two possible fates: B  C  DA H  I  J • If we have a lot of J around, it will bind to the enzyme that converts A to B and activate it; that will balance D with J! Biochemistry: Protein Function

  14. How does this work structurally? • In general, binding of the allosteric effector causes a medium-sized (~2-5Å) shift in the conformation of the protein • This in turn alters its properties • Affinity for the ligand • Flexibility (R vs T) • Other properties • We’ll revisit this when we do enzymology Biochemistry: Protein Function

  15. Classes of proteins • Remainder of this lecture:small encyclopedia of theprotein functions • Be aware of the fact thatproteins can take onmore than one function • A protein may evolve for one purpose • … then it gets co-opted for another • Moonlighting proteins (Jeffery et al, Tobeck) Arginosuccinate lyase / Delta crystallinPDB 1auw, 2.5Å206kDa tetramer Biochemistry: Protein Function

  16. Structural proteins • Perform mechanical or scaffolding tasks • Not involved in chemistry, unless you consider this to be 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 Biochemistry: Protein Function

  17. 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 PDB 2vb10.65Å, 14.2kDa Biochemistry: Protein Function

  18. Many enzymes are oligomeric • Both heterooligomers and homooligomers • ADH: tetramer of identical subunits • RuBisCO: 8 identical large subunits, 8 identical small subunits PDB 2hcy: tetramer PDB 1ej7: 2.45Å8*(13.5+52.2kDa) Biochemistry: Protein Function

  19. IUBMB Major Enzyme Classes Biochemistry: Protein Function

  20. Electron-transport proteins • Involved in Oxidation-reductionreactions via • Incorporated metal ions • Small organic moieties (NAD, FAD) • Generally not enzymes because they’re ultimately altered by the reactions in which they participate • But they can be considered to participate in larger enzyme complexes than can restore them to their original state Recombinant human cytochrome cPDB 1J3SNMR structure11.4kDa Biochemistry: Protein Function

  21. Sizes and characteristics • Some ET proteins: fairly small • Cytochrome c • Some flavodoxins • Others are multi-polypeptide complexes • Cofactors or metals may be closely associated (covalent in cytochromes) or more loosely bound Anacystisflavodoxin PDB 1czn1.7Å18.6 kDa Biochemistry: Protein Function

  22. Storage and transport proteins • Hemoglobin, myoglobin classic examples • “honorary enzymes”: share some characteristics with enzymes • Sizes vary widely • 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! Sperm-whale myoglobin Biochemistry: Protein Function

  23. Why do we have storage proteins? • Many metabolites are toxic in the wrong places or at the wrong times • Oxygen is nasty • Too much Ca2+ or Fe3+ can be hazardous • So storage proteins provide ways of encapsulating small molecules until they’re needed; then they’re released T.maritimaferritinPDB 1z4a8*(18 kDa) Biochemistry: Protein Function

  24. Hormones • Transported signaling molecules,secreted by one tissue and detectedby receptors in another tissue • Signal noted by the receptor will trigger some kind of response in the second tissue. • They’re involved in cell-cell or tissue-to-tissue communication. • Not all hormones are proteins • some are organic, non-peptidic moieties • Others: peptide oligomers, too small to be proteins • But some hormones are in fact normal-sized proteins. Human insulinPDB 1t1k 3.3+2.3 kDa Biochemistry: Protein Function

  25. Receptors • Many kinds, as distinguished by what they bind: • Some bind hormones, others metabolites, others non-hormonal proteins • Usually membrane-associated: • a soluble piece sticking out • Hydrophobic piece in the membrane • sometimes another piece on the other side of the membrane • Membrane part often helical:usually odd # of spanning helices (7?) Retinal from bacteriorhodopsinPDB 1r2nNMR structure27.4 kDa Biochemistry: Protein Function

  26. Why should it work this way? • Two aqueous domains, one near N terminus and the other near the C terminus, are separated by an odd number of helices • This puts them on opposite sides of the membrane! Biochemistry: Protein Function

  27. Nucleic-acid binding proteins • Many enzymes interact with RNA or DNA • But there are non-catalytic proteins that also bind nucleic acids • Scaffolding for ribosomal activity • Help form molecular machines for replication, transcription, RNA processing: • These often involve interactions with specific bases, not just general feel-good interactions • Describe these as “recognition steps” Human hDim1PDB 1pqnNMR struct.14kDa Biochemistry: Protein Function

  28. Scaffolding(adapter) proteins Human regulatory complex(Crk SH2 + Abl SH3)PDB 1JU5NMR structure • A type of signaling protein(like hormones and receptors) • Specific modules of the protein recognize and bind other proteins:protein-protein interactions • They thereby function as scaffolds on which a set of other proteins can attach and work together Biochemistry: Protein Function

  29. Protective proteins E5 Fragment of bovine fibrinogenPDB 1JY2, 1.4Å2*(5.3+6.2+5.8) kDa • Eukaryotic protective proteins: • Immunoglobulins • Blood-clotting proteins(activated by proteolytic cleavage) • Antifreeze proteins Biochemistry: Protein Function

  30. Other protective and exploitive proteins Vibrio cholerae toxin A1 + ARF6PDB 2A5F2.1Å21.2+19.3 kDa • Plant, bacterial, and snake-venom toxins • Ricin, abrin (plant proteins that discourage predation by herbivores) Synthetic Abrin-APDB 1ABR2.14Å29.3+27.6 kDa Biochemistry: Protein Function

  31. Special functions Dioscoreophyllum Monellin PDB 1KRL5.5+4.8 kDa • Monellin: sweet protein • Resilin: ultra-elastic insect wing protein • Glue proteins (barnacles, mussels) • Adhesive ability derived from DOPA crosslinks • Potential use in wound closure! Biochemistry: Protein Function

  32. What percentages do what? • See fig. 5.32 in G&G • 42% of all human proteins have unknown function! • Enzymes are about 20% of proteins with known functions (incl. 3% kinases, 7.5% nucleic acid enzymes) • Structural proteins 4.2% • Percentages here reflect diversity, not mass Biochemistry: Protein Function

  33. Fig.15 from Venter et al. (2001), Science 291:1304 Protein Functions Biochemistry: Protein Function

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