1. Protein Folding & Biospectroscopy F14PFB David Robinson Mark Searle Jon McMaster http://robinson.chem.nottingham.ac.uk/teaching

2. Module Overview The course will develop an understanding of protein structure, stability, design and methods of structural analysis; understand the protein folding problem and experimental approaches to the analysis of protein folding kinetics and the application of site-directed mutagenesis. A range of experimental spectroscopic techniques will be introduced to probe protein structure and stability based on secondary structure and tertiary interactions and to probe the nature of the active site of metalloproteins using equilibrium and time-resolved spectroscopy.

3. Protein Folding 1.Introduction 2.Protein Structure 3.Interactions 4.Protein Folding Models 5.Biomolecular Modelling 6.Bioinformatics Handouts: http://robinson.chem.nottingham.ac.uk/teaching/F14PFB

5. 3D Structure of Myoglobin - first to be determined by x-ray crystallography - revealed how the protein bound heme (loaded with oxygen) and gave the first detailed look at a protein structure - now 10,000’s of protein structures are known

6. The many functions of proteins  Mechanoenzymes: myosin, actin  Rhodopsin: allows vision  Globins: transport oxygen  Antibodies: immune system  Enzymes: pepsin, renin, carboxypeptidase A  Receptors: transmit messages through membranes And hundreds of thousands more…

7. Proteins are chains of amino acids  Polymer – a molecule composed of repeating units

8. Amino acid composition  Basic Amino Acid Structure: The side chain, R, varies for each of the 20 amino acids CR CC H N O OH H H Amino group Carboxyl group Side chain

9. The Peptide Bond  Dehydration synthesis  Repeating backbone: N–C  –C –N–C  –C Convention – start at amino terminus and proceed to carboxy terminus OO

10. Peptidyl polymers  A few amino acids in a chain are called a polypeptide. A protein is usually composed of 50 to 400+ amino acids.  Since part of the amino acid is lost during dehydration synthesis, we call the units of a protein amino acid residues. carbonyl carbon amide nitrogen

11. Side chain properties  Recall that the electronegativity of carbon is at about the middle of the scale for light elements Carbon does not make hydrogen bonds with water easily – hydrophobic O and N are generally more likely than C to h-bond to water – hydrophilic  We group the amino acids into three general groups: Hydrophobic Charged (positive/basic & negative/acidic) Polar

12. The Hydrophobic Amino Acids Proline severely limits allowable conformations!

13. The Charged Amino Acids

14. The Polar Amino Acids

15. More Polar Amino Acids And then there’s…

16. Amino acids

17. Planarity of the peptide bond Phi (  ) – the angle of rotation about the N-C  bond. Psi (  ) – the angle of rotation about the C  -C bond. The planar bond angles and bond lengths are fixed.

18. Phi and psi  =  = 180° is extended conformation  : C  to N–H  : C=O to C  CC C=O N–H

19. The Ramachandran Plot  G. N. Ramachandran – first calculations of sterically allowed regions of phi and psi  Note the structural importance of glycine Observed (non-glycine) Observed (glycine) Calculated

20. Four levels of protein structure Primary: amino acid sequence Ser Val Tyr Cys

21. Four levels of protein structure Primary: amino acid sequence Secondary: regular, repeated coiling and folding of polypeptide backbone

22. Four levels of protein structure Primary: amino acid sequence Secondary: regular, repeated coiling and folding of polypeptide backbone Tertiary: complete three-dimensional structure Quaternary: arrangement of subunits (in multisubunit protein)

23. Secondary structure Regular, repeated coiling and folding of polypeptide backbone  Due to hydrogen bonding  Two patterns  (alpha) helix  (beta) sheet

24. Tertiary  Complete three-dimensional structure  Due to weak interactions between side (R) groups as well as covalent disulfide bonds Weak interactions Hydrogen bonds Electrostatic interactions (ionic bonds) Hydrophobic interactions Van der Waals interactions

25. Tertiary structure formed through side chain interactions

27. Tertiary  Complete three-dimensional structure  Composed of: Motifs: specific combinations of secondary structural elements Domains: structurally independent units

28. Motifs specific combinations of secondary structural elements

29. Domains Structurally independent units Two different binding domains to bind two different molecules

30. Tertiary  Complete three-dimensional structure Native conformation: functional structure Most stable conformation

31. Tertiary Fibrous Proteins = extended filaments or Globular proteins = compact folded structure