Proteins

1. Proteins AHMP 5406

2. Objectives • Discuss the shape and structure of proteins. • List some different functions of proteins. • Describe the process of protein binding to another molecule. • Discuss the class of proteins called enzymes and their catalytic functions. • Explain the different ways in which the catalytic activities of enzymes are regulated. • Discuss the function of proteins as cellular regulators. • Discuss the function of motor proteins and membrane-bound transporter proteins.

3. Structure of Proteins • Def – a long chain of amino acids linked by covalent peptide bonds • AKA polypeptides • Shape is determined by amino acid sequence • Polypeptide backbone forms a repetitive core • Characteristics of a protein are due to their amino acid side chains

4. Amino acid structure

5. AA side chain characteristics • Can be • Polar • Negatively charged - Acidic • Positively charged - Basic • Uncharged but still polar • Nonpolar • hydrophobic

6. Shape of Proteins • The final folded structure of a protein is referred to as its conformation • Unfolded proteins are denatured • Fold according to lowest energy • Three types of bonds determine protein conformation • Ionic bonds • Hydrogen bonds • Van der Waals attractions

7. Interactions in aqueous environments • Polar side chains interact with water • Nonpolar (hydrophobic) side chains interact with each other

8. Hydrogen bonds • Help stabilize protein conformation

9. Common Folding Patterns • a helix • Cell membrane proteins • Formed when polypeptide chains twist around themselves • b sheet • Formed when chains run parallel to each other

10. Organizational Units • Four structural levels • Primary – AA sequence • Secondary – a helices or b sheets • Tertiary – 3D shape of pp chain • Quaternary – complex of more than one pp chain • Protein domain is the fundamental unit • Substructure that can fold independently into a compact and stable structure

11. Selection and protein structure • Few of large # of polypeptide chains will be useful • Typical protein is approx. 300 AA in length • Given 20 AA we can have 20300 possible polypeptide chains • Only a small fraction would be stable • Natural selection eliminates proteins that are unstable and have unpredictable biochemical properties

12. Protein Families • Evolutionarily successful proteins can be modified or duplicated • Allows for novel functions • Structurally related proteins can be classified into protein families • E.g. serine proteases include • Chymotrypsin, trypsin, elastase and blood clotting proteases • Protein families are recognized by DNA sequence similarities

13. Domain Shuffling • Multidomain proteins most likely originated from the joining of DNA sequences that code for each domain • Referred to as domain shuffling • Allow for novel combinations and functions • Particularly mobile domains are called protein modules

14. Protein Modules • Relatively small, 40-200 AA • Have versatile structures • Can be easily integrated into other proteins

15. Large proteins contain multiple polypeptide chains • Weak noncovalent bonds allow proteins to bind to each other • Binding site is area of interaction between protein and another molecule (or protein) • Thus protein subunits can form larger proteins

16. Protein structures and uses • Some proteins from long helical filaments • Identical protein subunits have complementary binding sites on either end • Depending on position of binding site can also form rings • Actin found in the cytoskeleton

17. Protein structures and uses • Fibrous protein molecules • Elongated three-dimensional structures • Used for spanning relatively long distances in the cell • a-keratin formed by two a-helices • Coiled coil • Intermediate filaments • Cytoskeleton • Extracellular matrix of cells • Collagen • Triple helix • Elastin found in resilient tissues

18. Extracellular proteins • Stabilized by covalent cross-linkages • Bind two amino acids • Connect different polypeptide chains • Most common are disulfide bonds • Formed as proteins are being modified for export • Endoplasmic reticulum

19. Proteins can be part of supramolecular structures • Same binding mechanisms can allow proteins to form larger structures • E.g. enzyme complexes, ribosomes, protein filaments, viruses, and membranes • Multiple protein contacts adds stability to overall structure

20. Large protein assembly • Assembly on a core • a protein core of macromolecule provides scaffold • protein length determined by the core length • TMV tail length determined by RNA chain • Thin filaments in muscle

21. Large protein assembly • Accumulated Strain • Assembly is terminated due to strain of polymer • Addition of subunits becomes energetically unfavorable • Vernier Mechanism • Vernier scale • Molecules based on differently sized subunits grow until their ends match

22. Protein Function

23. Intro to protein binding • Biological properties of proteins depends on interaction with other molecules • Binding is usually very specific • A ligand is a substance that binds to a protein by noncovalent bonds • Ion • Small molecule • Macromolecule • Ligand binds to binding site

24. Protein conformation = chemical reactivity • Binding site modification • Folding can create specific binding sites • Ex. Water can compete with ligand • Clustering nonpolar AA’s will repel water • Or activate nonreactive AA’s

25. Protein conformation = chemical reactivity • Reactive amino acids at binding site • Ex. If you put many neg side chains together • Then you increase affinity for positively charged ligand • Can change reactivity of unreactive side group

26. Types of binding interfaces • Surface-string • Protein contacts extended loop of another protein’s polypeptide chain • Ex. SH2 domain can recognize phosphorylated polypeptide loop on other proteins • Helix-Helix • AKA coiled-coil • Surface-surface • Most common • Tight interactions because many weak bonds • Very specific

27. Antibody binding sites • Antibodies proteins produced by immune system in response to foreign molecules • Antibodies bind to a target called antigen • Have two identical binding sites (Y) • Formed by many loops • Which are good for binding • B/C many chemical groups can surround the ligand

28. Enzymes • Make and break covalent bonds • Bind to ligands called substrates • Convert substrates into products • Can also speed up reactions – catalysts • Enzymes are grouped into classes by type of reaction

29. Enzyme Classes • Hydrolases – hydrolytic cleavage • Nucleases – break nucleotide bonds • Proteases – break amino acid bonds • Phosphatases – remove phosphate groups • ATPases – hydrolyze ATP • Synthases – synthesize molecules by anabolic reactions • Isomerases – rearrange bonds within molecules • Polymerases – synthesize DNA and RNA • Kinases – add phosphate groups to molecules • Oxido-Reductases – one molecule is oxidized another is reduced

30. Substrate binding • First step in enzyme catalysis • E = enzyme • S = substrate • P = product • Enzymes can be reused E + S  ES  EP  E + P

31. Enzymes can speed up reactions • By stabilizing transition states of substrates • Concentrating substrate molecules at binding sites • Binding energy contributes to catalysis • Decreases activation energy

32. Example of rate accelerations

33. Small molecules add functionality • Non protein molecules can allow difficult reactions to occur • Hemoglobin • Four heme groups and iron atom • Carboxypeptidase • Cuts polypeptide chains • Has zinc in its active site to assist reaction • Coenzymes – organic molecules • Vitamins – can not be produced by cell

34. Regulation of catalytic activities • Expression of gene • Compartmentalization • Targeted degradation of enzymes • Another molecule binds to a regulatory site • Affects rate of reaction • Feedback inhibition • Enzyme early in the reaction is affected by later product • AKA – negative regulation • Positive regulation • Later product reinforces enzyme activity

35. Allosteric enzymes • Have two binding sites • Active site • Regulatory site • Binding of regulatory site can lead to conformational changes • Affects rate of reaction

36. Enzyme Linkage • One ligand can affect binding of another ligand • Due to conformation change • These two sites are coupled • Can increase or decrease affinities for ligands.

37. Phosphorylation • Regulates enzyme function • Causes conformational changes • Phosphate can contribute to binding site • Phosphorylation of enzymes can be signaled • External stimulus • Protein kinases

38. Protein kinases and phosphatases • Proteins are phosphorylated by addition of terminal P group from ATP • Serine, Threonine or Tyrosine • Reaction catalyzed by protein kinase • Unidirectional due to energy release

39. Protein kinases and phosphatases • Protein phosphatases • Dephosphorylate proteins • Remove phosphate group

40. Proteins as cellular regulators • Signaling devices • Cdk • controls cell cycles • only active if bound to cyclin and phosphate • and if also simultaneously dephosphorylated somewhere else • Src another example • signal intergrators

41. Proteins as cellular regulators

42. GTPases • Bound GTP is hydrolyzed • Causes conformational change • Activates protein • Controlled by regulatory proteins • GAP – GTPase activating protein • GEF – Guanine nucleotide exchange factor

43. Elongation Factors • Some proteins required as assembly factors • E.g. Building polymers • EF-Tu protein • Elongation factor in protein synthesis • Loads each amino-acyl tRNA molecule onto ribosome • tRNA bound and masked by EF-TU when GTP is present • If bound to ribosome and phosphate group is hydrolyzed (GTP  GDP) • tRNA is unmasked • tRNA transfers AA to the polypeptide

44. Motor Proteins • Function to move other proteins • Muscle contractions • Chromosome movement • Organellar movement • Work by unidirectional conformation changes • By coupling a change with ATP hydrolysis • Energetically unfavorable to go in reverse

45. Membrane Transport Proteins • Harness energy • ATP hydrolysis • Ion gradients • Electron transport processes • Ex. Ca2+ pump • Muscle cells • Specialized organelle – sarcoplasmic reticulum • Pumps Ca ions out of cell to maintain low levels • ATP hydrolysis drives conformation change