1. Protein Folding & Biospectroscopy F14PFB Dr David Robinson Lecture 2

2. Principles of protein structure and function Function is derived from structure Structure is derived from amino acid sequence Different activities and shapes of proteins due to different amino acid sequences

3. A reminder… 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

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

5. Levels of Protein Structure  - helix Myoglobin Hemoglobin The folded protein structure is stabilized by a variety of weak chemical interaction, and in some cases covalent (disulfide) bonds between cysteine residues R– CH 2 –S–S–CH 2 –R Cys Disulfide bond:

6. Structural elementDescription primary structureamino acid sequence of protein secondary structurehelices, sheets, turns/loops super-secondary structureassociation of secondary structures domainself-contained structural unit tertiary structurefolded structure of whole protein includes disulfide bonds quaternary structureassembled complex (oligomer) homo-oligomeric (1 protein type) hetero-oligomeric (>1 type) Protein structure: overview

7. Primary & Secondary Structure  Primary structure  Primary structure = the linear sequence of amino acids comprising a protein: AGVGTVPMTAYGNDIQYYGQVT…  Secondary structure Regular patterns of hydrogen bonding in proteins result in two patterns that emerge in nearly every protein structure known: the  -helix and the  -sheet secondary structureThe location of direction of these periodic, repeating structures is known as the secondary structure of the protein

8. The alpha helix      60°

9. Properties of the alpha helix      60°  Hydrogen bonds  Hydrogen bonds between C=O of residue n, and NH of residue n+4  3.6 residues/turn  1.5 Å/residue rise  100°/residue turn

10. Properties of  -helices  4 – 40+ residues in length  Often amphipathic or “dual-natured” Half hydrophobic and half hydrophilic Mostly when surface-exposed  If we examine many  -helices, we find trends… Helix formers: Ala, Glu, Leu, Met Helix breakers: Pro, Gly, Tyr, Ser

11. The beta strand (& sheet)    135°   +135°

12. Properties of beta sheets  Formed of stretches of 5-10 residues in extended conformation  Pleated – each C  a bit above or below the previous  Parallel/antiparallel  Parallel/antiparallel, contiguous/non-contiguous

13. Parallel and anti-parallel  -sheets  Anti-parallel is slightly energetically favoured Anti-parallelParallel

14. Turns and Loops  Secondary structure elements are connected by regions of turns and loops  Turns – short regions of non- , non-  conformation  Loops – larger stretches with no secondary structure. Often disordered. “Random coil” Sequences vary much more than secondary structure regions

15. Levels of Protein Structure  Secondary structure elements combine to form tertiary structure  Quaternary structure occurs in multienzyme complexes Many proteins are active only as homodimers, homotetramers, etc.

16. Protein Folding Forming polypeptide chain requires energy and information (template) – ie translation from RNA  protein SEQUENCE

17. Protein Folding Forming polypeptide sequence requires energy and information (template) Forming native conformation requires NO ADDITIONAL energy or information (SELF ASSEMBLY)

18. Protein folding Amino acid sequence contains all information necessary for folding into a specific three- dimensional structure

19. Protein Folding Proteins, in general, do NOT fold as they are synthesized on the ribosome

20. Folding of RNAse A in the test tube denaturationrenaturation Incubate protein in guanidine hydrochloride (GuHCl) or urea 100-fold dilution of protein into physiological buffer Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230. - the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors”

21. Protein Folding Many proteins fold by Assisted Self Assembly Correct assembly (native conformation) requires assistance by CHAPERONES

22. Protein unfolding = Denaturation Loss of structure and function –Heat –Extreme pH –Detergents –Urea

23. Protein unfolding = Denaturation Why do these conditions cause loss of structure and function? –Heat –Extreme pH –Detergents –Urea

24. Lysozyme

26. Tertiary: complete three-dimensional structure Quaternary: arrangement of subunits (in multisubunit protein)

27. Hemoglobin

28. Quaternary structure Held together by weak interactions between side (R/functional) groups as well as covalent disulfide bonds

29. Structure-function relationship Function is derived from structure Structure is derived from sequence

30. Sickle-cell disease Normal red blood cells Sickle shaped red blood cells Due to single amino acid change in haemoglobin

31. Sickle-cell disease

32. Single specific amino acid change causes change in protein structure and solubility Results in change in cell shape Causes cells to clog blood vessels

33. Amino acids