Chapter 3 Proteins:

1. Chapter 3 Proteins: Shape, Structure, and Function

2. Proteins Execute Cell Functions • Enzymes • Channels and pumps • Signal Molecules • Messengers • Molecular Machines • Structural Support • Cell Recognition

3. Protein Shape and Structure Peptide Bond Links Amino Acids into Polypeptide Chain

4. Protein Shape and Structure • Evolution fine-tuned structure and chemistry • Shape dictated by amino acid sequence polypeptide backbone side chains

5. Protein Shape and Structure Sequence Determines Structure

6. Protein Shape and Structure Weak Noncovalent Bonds/Interactions important to the folding of polypeptide chain

7. Protein Shape and Structure • Fold into Conformation of Lowest Energy • Common Folding Patterns alpha helix Beta Sheet Coiled Coils

8. Protein Shape and Structure Levels of organization protein structure • primary= aa seqeunce • secondary= stretches of alpha helix, beta sheets • tertiary=3d organization • quartenary=complete structure of protein w/ > 1 poly-peptide chain

9. Protein Shape and Structure • Protein Domain= Fundamental Unit of Organization independently folding unit 40-350 aa modular unit; combine to form larger proteins different domains have different functions • Fold= central core of domain; comprised of beta sheets and alpha helices in various combinations; limited number Short signature sequences identify homologous protein domains

10. Protein Shape and Structure Domain shuffling during the course of evolution Percentage of total genes in respective genomes containing one or more copies of a particular protein domain

11. Protein Shape and Structure Protein Module • Smaller than an average domain, generally 40-200 aa • Particular versatile structures • Easily integrated into other proteins; form parts of many different proteins

12. Protein Shape and Structure Protein Families Evolved • similar 3d structure • portions or aa sequence conserved • non-conserved portions impart new functionality serine proteases homeodomain proteins kinases immunoglobulins

13. Protein Shape and Structure • Sequence Homology Searches • Amino Acids Sequence Threading • Modules form parts of many different proteins

14. Protein Shape and Structure

15. Protein Shape and Structure Larger proteins can assemble from identical monomeric subunits

16. Protein Shape and Structure • Larger proteins often contain more than one polypeptide • Proteins can serve as subunits for assembly of large structures • Self Assembly

17. Protein Function • Function of protein dictated by physical interactions w/ other molecules specificity and ligand affinity governed by multiple weak noncovalent bonds active/binding site often cavity on protein surface formed by neighboring aa or aa that may belong to different portions of polypeptide

18. Protein Function Conformation determines chemistry • Regions adjacent to active or ligand binding site may restrict water to increase ligand binding • Clustering of polar or chged residues can alter chemical reactivity • Type and orientation of exposed aa side chains govern chemical reactivity

19. Protein Function “Evolutionary tracing” to determine sites critical to protein function • 3d structure of protein family members are similar even when aa homology falls to 25% • Map unchg aa or nearly unchg from all known family members onto 3d structure of one family member • Most invariant positions often on surface and represent ligand binding site

20. Protein Function Proteins Bind to other Protein Through Several Types of Interfaces

21. Protein Function Equilibrium Constant Describes Binding Strength • Steady state or equilibrium: # association events/sec = # dissociation/sec • From conc of two molecules and complex equilibrium constant can be calculated

22. Protein Function Enzymes as Catalysts • Make or break covalent bonds • Speed up chemical reactions > 106 fold • Stabilize transition state • Decrease activation energy • Increase local conc of substrate at catalytic site • Hold reactants in proper orientation for chem rxn • Binding energy contributes directly to catalysis • Not consumed or changed during process

23. Protein Function Common Types of Enzymes Hydrolases Isomerases OxidoReductases Nuclease Polymerases ATPases Proteases Kinases Synthases Phosphatases

24. Protein Function Enzyme Kinetics • Vmax= how fast enzyme can process substrate, pt at which enzyme saturated w/substrate • Turnover Number= Vmax/[enzyme] turnover ranges from 1-10,000 substrate molec/sec • Km= substrate conc at Vmax/2; measure of affinity

25. Protein Function Lysozyme • Natural antibiotic in egg white, tears, saliva • Hydrolyzes polysaccharide chains residing in cell wall of bacteria

26. Protein Function Specific Mechanism of Lysozyme Hydrolysis • Enzyme positions substrate bending critical chem bonds that participate in chem rxn • Positions acidic side chain of Glu w/in active site to provide high conc of acidifying H+ ions • Negatively chged Asp stabilizes positive chged transition state

27. Protein Function General Mechanism for Enzyme Activity • Active site contains atoms that speed up rxn • Substrate driven towards transition state upon binding to enzyme; shape of substrate chgs & critical bonds bent • Covalent bond sometimes formed btwn substrate and side chain of enzyme • Restoration of side chain to original state

28. Protein Function Small Molecules Add Extra Functions to Proteins • Chromophores detect light; retinal • Metal atoms assist w/ catalytic functions; Zn, Mg, Fe • Coenzymes (sm organic molec) provide functional grps; biotin

29. Protein Function Multienzyme Complexes • Increase the rate of cell metabolism • Product of enzyme A passed directly to enzyme B; product of enzyme B passed to enzyme C; and so on • Simulates intracellular membrane compartment; effectively increasing substrate conc at site of enzyme activity

30. Protein Function Regulation of Catalytic Activity • Negative Feedback • Positive Regulation • Allosterism

31. Protein Function Allosterism

32. Protein Function Symmetric Protein Assemblies and Cooperative Allosterism: sm chgs in ligand conc switches enzyme assembly from fully active to fully inactive state via conformation changes that are transmitted across neighboring subunits

33. Protein Function Allosteric Transition in Aspartate Transcarbamoylase • 6 catalytic subunits and 6 regulatory subunits • all or none transition between T-tense and R-relaxed state • Active R state driven by binding of carbamoylphosphate and aspartate • Inactive T state driven by binding of CTP to regulatory dimers

34. Protein Function Regulation by Phosphorylation/Dephosphorylation • Addition or removal of P grp carrying (2) negative chgs can cause major conformation chg in protein • Phosphorylation/dephosphorylation of proteins= response to signals that specify chg in cell state

35. Protein Function Protein Kinase • transfers terminal P of ATP to OH grp of SER, Thr, or Tyr • 100’s ea specific for particular target • Kinases share 250 aa catalytic domain • Non-conserved aa flanking catalytic site or in loops w/in kinase domain confer specificity

36. Protein Function Protein Phosphatases • Catalyzes the removal of P grp • Some specific; some act on broad range of proteins

37. Protein Function Protein can Function as Microchip Cdk= cyclin dependent protein kinase activity dependent upon 3 events: 1. binding of second protein cyclin 2. phosphorylation of Thr side chain 3. dephosphorylation of Tyr side chain Cdk monitors specific set of cell components acting as input-output device

38. Protein Function GTP Binding Proteins • Analogous to Proteins regulated by P/de-P • Active when GTP bound; inactive when GTP hydrolyzed

39. Protein Function Regulatory Proteins Control Activity of GTP Binding Proteins • GAP= GTPase activating protein; binds and induces hydrolysis • GEF= Guanine nucleotide exchange factor; binds to GDP protein causing it to release GDP in exchange for GAP

40. Protein Function Large Protein Movements Generated from Small Ones • EF-Tu = elongation factor in protein synthesis, GTPase 1. tRNA complexes w/ GTP bound form of EF-Tu w/ aa masked 2. GTP hydrolysis occurs when tRNA binds to mRNA on ribosome; tRNA disassociates 3. GTP hydrolysis causes “Swtich helix” to swivel unmasking aa

41. Protein Function Motor Proteins • Produce lg movements in cells such as: muscle contraction crawling and swimming of cells movement of chromosomes movement of organelles enzymes on DNA • Possess moving parts as force generating machines

42. Protein Function ATP hydrolysis allows unidirectional series of conformational chgs to propel proteins along DNA

43. Protein Function Allosteric proteins harness energy derived from ATP hydrolysis, ion gradients, electron transport processes to pump ions or sm molecules across membranes Ca2+ Pump of Sarcoplasmic Reticulum

44. Protein Function Mechanism of Ca2+ Pump

45. Protein Function Structure of Ca2+ Pump • 10 transmembrane helices • 4 transmembrane helices provide Ca2+ binding sites for pump • helices that bind Ca2+ wind around ea other forming cavity btwn helices for Ca2+ • ATP hydrolysis causes conformation chgs that later cavity enabling Ca2+ to be pushed through