1. Chapter 7 Protein Function Ligand --- a molecule bound reversibly by a protein Binding site --- the site on protein to which a ligand binds Induced fit --- the structure adaptation that occurs between protein and ligand Substrate --- the molecule acted upon by enzymes Catalytic/active site --- the substrate/ligand binding site Ch 7 --- Noncatalytic functions of proteins

2. Fig. 7-1,2 Heme Reversible binding of a protein to a ligand: Oxygen-binding proteins protoporphyrin

3. Fig. 7-3 The structure of myoglobulin *a single binding site for O 2 *78%  helices (8) *His 93 or HisF8 (the 8 th residue in  helix F) binds to heme *Bends between  helices

4. Protein-ligand interactions can be described quantitatively P + L PL Ka = [PL]/[P][L] Kd = [P][L]/[PL]  = (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka) Fig. 7-4 Graphical representation of ligand binding  = 0.5 [L] = 1/Ka, or Kd

5. Dissociation constant, K d = [P][L]/[PL] P + L PL Ka = [PL]/[P][L]  = (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka) = [L]/([L] +Kd) When [L] = Kd  = 0.5 (half saturation) [L] = 9 Kd  = 0.9 Kd: the molar concentration of ligand at which half of the available ligand-binding sites are occupied Kd, affinity ( ? )

7. When O 2 binds to Mb P 50 = 0.26 kPa  = [L]/([L] +Kd) = [O 2 ]/([O 2 ] + Kd) = [O 2 ]/([O 2 ] + [O 2 ] 0.5 ) The concentration of a volatile substance in solution is always proportional to its partial pressure in the gas phase above the solution  = pO 2 /(pO 2 + P 50 )

8. Protein structure affects how ligands bind 1. Steric effects 2. Molecular motions/breathing in the structure 1: 20,0001: 200

9. Oxygen is transported in blood by hemoglobin (Hb) In arterial blood, Hb ~96% saturated In venous blood, Hb ~64% saturated P 50 = 0.26 kPa Mb Mb has only one subunit, as an oxygen-storage protein

10. Fig. 7-6 Comparison between Mb and Hb 

11. Fig. 7-7 Comparison of aa between whale Mb and Hb ,  A-H helices Only 27 aa identical

12. Fig. 7-8 Dominant interactions between Hb subunits >30 aa 19 aa (hydrophobic, H-bonds, affected strongly upon O 2 binding)

13. Hb undergoes a structural change on binding oxygen Fig. 7-10 The T(tense) R(relaxed) transition

14. Fig. 7-9 Some ion pairs that stabilize the T state of deoxyHb

15. Fig. 7-11 Changes in conformation near heme on O 2 binding

16. Hb binds oxygen cooperatively Fig. 7-12 A sigmoid (cooperative) binding curve 4 vs. 13.3 kPa Mb – a single subunit protein Hb – 4 subunits, an allosteric protein

17. Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of another site on the same protein allos --- other stereos --- solid or shape Homotropic interaction --- liagnd = modulator Heterotropic interaction --- ligand = modulator O 2 --- as both a normal ligand and an activating homotropic modulator for Hb

18. Cooperative ligand binding can be described quantitatively Dissociation constant, Kd = [P][L] n /[PLn] P + nL PLn Ka = [PLn]/[P][L] n  = (binding sites occupied)/(total binding sites) = [L] n /([L] n +Kd)  = [L] n /Kd Log{  = n log [L] – log Kd (Hill equation, 1910) Log{  = n log pO 2 – log P 50 n H – the Hill coefficient (slope of Hill plot) 1 Fig. 7-13

19. Two models suggest mechanisms for cooperative binding Concerted (all-or-none), 1965 Sequential, 1966 Fig. 7-14

20. O 2 binding to Hb is regulated by 2,3-bisphosphoglycerate (BPG) HbBPG + O 2 HbO 2 +BPG 4 1 [BPG] during hypoxia Fig. 7-16

21. Fig. 7-17 Binding of BPG to deoxyHb T state T R O2O2 ++ BPG is negatively charged

22. Sickle-cell anemia is a molecular disease of Hb Val6 mutates to Glu6 in two  chains

23. Complementary interactions between proteins and ligands: The immune system and immunoglobulins MHC (major histocompatibility complex) all vertebrate cells macrophages, B cells

24. Structure of a human class I MHC protein Recognized by T-cell receptor

25. Over view of the immune response to a viral infection

26. The structure of immunoglobulin G (IgG)

27. Binding of IgG to an antigen

28. Induced fit in the binding of an antigen to IgG Heavy chain Light chain Kd~10 -10 M

29. The Ab-Ag interaction is the basis fro a variety of important analytical procedures Ployclonal vs. monoclonal Ab ELISA (enzyme-linked immunosorbent assay)

30. Immunoblot assay (Western Blot)

31. Protein interactions modulated by chemical energy Actin, myosin, and molecular motors Fig. 7-29 Myosin S1

32. The major components of muscle Fig. 7-29

33. Structure of skeletal muscle relaxed contracted

34. Muscle contraction

35. Molecular mechanism of muscle contraction 3~4 pN of forces, 5~10 nm movement/cycle