1 / 42

Protein Functions; Introduction to Carbohydrates

Protein Functions; Introduction to Carbohydrates. Andy Howard Introductory Biochemistry, Fall 2014 22 September 2014. Protein methods and functions. Today we ’ l l finish our discussion about how we learn about proteins Then we ’ l l comment on protein functions on a more systematic basis

barbaravaughan
Download Presentation

Protein Functions; Introduction to Carbohydrates

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein Functions; Introduction to Carbohydrates Andy HowardIntroductory Biochemistry, Fall 2014 22 September 2014 Protein Functions; Sugars

  2. Protein methods and functions • Today we’ll finish our discussion about how we learn about proteins • Then we’ll comment on protein functions on a more systematic basis • Finally, we’ll begin our exploration of carbohydrate chemistry Protein Functions; Sugars

  3. Structure methods Spectroscopy Scattering, MS Classes of proteins & their roles Structural Enzymes Electron transport Storage, transport Hormones Classes of proteins Receptors Nucleic-acid binding proteins Scaffolding & Adapter Protective Other Carbohydrates Definitions Properties Plans for Today Protein Functions; Sugars

  4. 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 (A = cl) • 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 Protein Functions; Sugars

  5. Solution scattering • Proteins in solution scatter X-rays in characteristic, spherically-averaged ways • Low-resolution structural information available • Does not require crystals • Until ~ 2000 you needed high [protein] • Thanks to BioCAT, SAXS on dilute proteins is becoming more feasible • Hypothesis-based analysis Protein Functions; Sugars

  6. Fiber Diffraction • Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering) • Produce characteristic fiber diffraction patterns • Collagen, muscle proteins, filamentous viruses Protein Functions; Sugars

  7. X-ray spectroscopy • All atoms absorb UV or X-rays at characteristic wavelengths • Higher Z means higher energy, lower for a particular edge • Perturbation of absorption spectra at E = Epeak +  yields neighbor information • Changes just below the peak yield oxidation-state information • X-ray relevant for metals, Se, I Protein Functions; Sugars

  8. Mass spectrometry as a structural tool • MS tells you molecular weights • Can give high precision in m/m • Not, strictly speaking, a way of determining structure • Can distinguish oligomeric state • Coupled with proteolytic digestion, it can be used to find interesting fragmentation patterns Protein Functions; Sugars

  9. Classes of proteins(G&G §5.8) • Reminder:proteins 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 Protein Functions; Sugars

  10. 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 Protein Functions; Sugars

  11. Enzymes(G&G §13.1) • 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 Protein Functions; Sugars

  12. 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) Protein Functions; Sugars

  13. IUBMB Major Enzyme Classes Protein Functions; Sugars

  14. Enzymes have 3 features • Catalytic power (they lower G‡) • Specificity • They prefer one substrate over others • Side reactions are minimized • Regulation • Can be sped up or slowed down by inhibitors and accelerators • Other control mechanisms exist Protein Functions; Sugars

  15. iClicker quiz, question 1 1. Collagen is a structural protein. Collagenase catalyzes the hydrolysis of collagen under appropriate circumstances. It is: • (a) an enzyme • (b) a hormone • (c) a receptor • (d) a nucleic-acid binding protein • (e) there’s no way to tell from the information provided. Protein Functions; Sugars

  16. iClicker quiz, question 2 2. Which IUBMB enzyme category would collagenase fall into? • (a) ligases (6) • (b) oxidoreductases (1) • (c) hydrolases (3) • (d) isomerases (5) • (e) none of the above. Protein Functions; Sugars

  17. iClicker quiz, question 3 • 3. Triosephosphateisomerase, whose structure we discussed last week, interconverts glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. What would you expect the approximate DG value for this reaction to be? • (a) -30 kJ mol-1 (d) 0 kJ mol-1 • (b) 30 kJ mol-1 (e) no way to tell. • (c) -14 kJ mol-1. Protein Functions; Sugars

  18. 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 Protein Functions; Sugars

  19. 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 Protein Functions; Sugars

  20. 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 myoglobinPDB 1MTJ 1.7Å 18kDa monomer Protein Functions; Sugars

  21. 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) Protein Functions; Sugars

  22. 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. Human insulinPDB 1t1k 3.3+2.3 kDa Protein Functions; Sugars

  23. Not all hormones are proteins • Some are organic, non-peptidic moieties • Others: peptide oligomers, too small to be proteins • Oxytocin: CYIQNCPLG • Angiotensin I: DRVYIHPFHL • Some are cyclic (COO- terminus and NH3+ termini hydrolytically ligated) • But some hormones are in fact normal-sized proteins. Protein Functions; Sugars

  24. 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 Protein Functions; Sugars

  25. 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! Protein Functions; Sugars

  26. Nucleic-acid binding proteins • Many enzymes interact with RNA or DNA • But there are non-catalytic proteins that also bind nucleic acids Human hDim1PDB 1pqnNMR struct.14kDa Protein Functions; Sugars

  27. Non-catalytic nucleic acid binding proteins • 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” Protein Functions; Sugars

  28. Non-catalytic nucleic acid binding proteins • 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” Protein Functions; Sugars

  29. Scaffolding(adapter) proteins • 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 Human regulatory complex(Crk SH2 + Abl SH3)PDB 1JU5NMR structure Protein Functions; Sugars

  30. 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 Protein Functions; Sugars

  31. 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 Protein Functions; Sugars

  32. 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! L-DOPA Protein Functions; Sugars

  33. What percentages do what? • 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 Protein Functions; Sugars

  34. Fig.15 from Venter et al. (2001), Science291:1304; G&G Fig. 5.26 Protein Functions Protein Functions; Sugars

  35. iClicker quiz, question 4 • 4. Suppose a membrane protein has 4 transmembrane helices and 2 aqueous domains, one at the N-terminal end and the other at the C-terminal end. Assuming the N-terminal aqueous domain is in the cytoplasm, where would the C-terminal aqueous domain be?(a) in the cytoplasm(b) in the membrane(c) in the extracellular space(d) no way to tell Protein Functions; Sugars

  36. Carbohydrates(G&G Chapter 7) • Sugars are vital as energy sources, and they also serve as building blocks for lipid-carbohydrate and protein-carbohydrate complexes. These are the most abundant organic molecules on the planet, and they act as metabolites, components of complexes, and structural entities. Protein Functions; Sugars

  37. Carbohydrates • These are polyhydroxylated aldehydes and ketones, many of which can exist in cyclic forms • General monomeric formula (CH2O)m, 3 m 9 • With one exception (dihydroxyacetone) they contain chiral centers • Monomers and small oligomers: highly soluble • Can be oligomerized and polymerized • Oligomers may or may not be soluble • Most abundant organic molecules on the planet Protein Functions; Sugars

  38. Aldoses & ketoses(G&G §7.2) • If the carbonyl moiety is at the end carbon (conventionally counted as 1), it’s an aldose • If carbonyl is one carbon away (counted as 2), it’s a ketose • If it’s two or more carbons from the end of the chain, it’s not a sugar Protein Functions; Sugars

  39. Simplest monosaccharides • Glyceraldehyde and dihydroxyacetone • Only glyceraldehyde is chiral:D-enantiomer is more plentiful in biosphere • All longer sugars can be regarded as being built up by adding-(CHOH)m-1 to either glyceraldehyde or dihydroxyacetone, just below C2 Protein Functions; Sugars

  40. How many aldoses are there? • Every -(CHOH) in the interior offers one chiral center • An m-carbon aldose has (m-2) internal -(CHOH) groups • Therefore: 2m-2 aldoses of length m • For m=3, that’s 21=2; for m=6, it’s 24=16. Protein Functions; Sugars

  41. How many ketoses are there? • Every -(CHOH) in the interior offers one chiral center • An m-carbon ketose has (m-3) internal-(CHOH) groups • Therefore: 2m-3 ketoses of length m • For m=3, that’s 20 = 1; for m=6, that’s 23=8. Protein Functions; Sugars

  42. Review: stereochemical nomenclature • Stereoisomers: compounds with identical covalent bonding apart from chiral connectivity • Enantiomers: compounds for which the opposite chirality applies at all chiral centers • Epimers: compounds that differ in chirality at exactly one chiral center • One chiral center: enantiomers are epimers. • > 1 chiral center: enantiomers are not epimers. Protein Functions; Sugars

More Related