Chapter 5: Protein Function

1. Chapter 5: Protein Function Dr. Clower Chem 4202

2. Functions of Proteins

3. Protein Function Very specific biological function Varies based on structure Fibrous proteins Enzymes Transport across membranes Common theme: how proteins bind to interact with other molecules Involves reversible binding with ligands (or substrates) in binding site (or active site) Can have multiple binding sites in one protein Binding may involve change in conformation (induced fit) Induced fit may cause change in other parts of the protein (e.g. other subunits) Interactions between ligands and proteins are regulated

4. Model Proteins Myoglobin and Hemoglobin Oxygen-binding proteins Transport Increase solubility of O2 in aqueous environment First 3D structures determined

5. Myoglobin Mb Transport protein Primarily in muscle tissue Structure Small, globular 153 residues 8 a-helices (A-H) connected by bends (AB, etc.) Contains one heme group Prosthetic group Binds in hydrophobic pocket

6. Heme Found in O2 transport proteins and electron-transfer proteins (cytochromes) Heterocyclic porphyrin ring system Conjugated Flat Binds divalent ion e.g. Fe2+ Located deep in protein structure to prevent oxidation of ion Between E and F helices Held in place by hydrophobic interactions (Phe, Val)

7. Heme Iron has 6 coordination bonds 4 to pyrrole groups 1 to His residue Proximal His F8; His93 1 to O2 or other small molecule (CO, NO)

8. Binding of Oxygen Equilibrium Ka = association constant Not the same as acid dissociation constant Measure of affinity of L for P Higher Ka = higher affinity Kd = dissociation constant Lower Kd = higher affinity Fractional saturation (q) Fraction of ligand-binding sites occupied by L

9. Kd = [L] at which � binding sites occupied Protein is half-saturated Lower Kd = smaller [L] required due to stronger binding between L and P

10. Reversible Binding of O2 to Mb Equilibrium expression Kd expression Substitute [O2] = pO2 Partial pressure Easier to measure Substitute Kd = [O2]0.5 Concentration at which � sites occupied P50 Expression for q

11. Plot of q vs. pO2 for Mb Low pO2 = low binding Increase pO2 = increased binding until saturated

12. Steric Factors O2 vs. CO binding Free heme vs. Mb heme Distal His = E7 (His 64)

13. Hemoglobin Hb Closely related to Mb More O2 transport Multiple subunits Multiple binding sites Responsiveness to changes in pO2

14. Hb Structure Spherical Tetrameric protein Quaternary structure = aabb a = 141 residues b = 146 residues Both a and b similar to Mb Structure, not sequence

15. Amino Acid Sequences of Mb and Hb

16. Hb Structure Dimer of ab protomers Rotational symmetry

17. Hb structure a and b units attract at interfaces a1b1 and a2b2 35 residues a2b1 and a1b2 19 residues Typically hydrophobic Also electrostatic and H-bonding a1a2 and b1b2 little or no interaction Separated by solvent channel Each subunit binds a heme Between E and F helices Heme binds O2 Structure changes when O2 binds

18. T State Deoxyhemoglobin Very little O2 affinity Some electrostatic interactions

19. R State Oxyhemoglobin Higher affinity of O2 ab dimer rotates ~15� a2b1 and a1b2 contacts shift b chains closer together Some ion pairs broken

20. O2 Binding to Hb pO2 higher in lungs than in tissue O2 needs to bind, then release This will not happen with Mb hyperbolic curve; animation Hb transitions T to R state as more O2 binds Cooperative interaction between binding sites Binding to one site affects binding to the other sites One O2 molecule binding increases O2 affinity of other sites Allosteric protein

21. Plot of q vs. pO2 for Hb Hb described by sigmoidal curve At low pO2, sites compete for first O2 ligand and weak binding in T state Slope increases quickly due to increased affinity of other sites (T ? R) Becomes saturated

22. Hill Equation Describes sigmoidal curve Expression for q n = number of binding sites Hill plot nH = slope = Hill coefficient Measure of degree of cooperativity (interaction between binding sites) Solving Hill equation shows that the 4th ligand binds with 100x greated affinity than 1st ligand

23. Hill Plot nH = 1 Hyperbola (like Mb) Ligand binding is not cooperative nH > 1 Positively cooperative (like Hb) Upper limit = n (4 for Hb; typical nH ~2.8-3.0; upper limit never reached) nH < 1 Negatively cooperative Reduce affinity when first ligand binds

24. Explanation for Cooperative Interaction Why does ligand affinity increase? How does one heme affect the others? Not through electronic mechanism Hemes too far apart (25 � 37 �) Due to change in structure upon oxygenation Perutz mechanism Change from T state conformation to R state conformation

25. Perutz Mechanism Very fast 1. Fe(II) in T state site above heme Fe(II) binds to O2 Fe(II) pulled down into heme (R state) 2. Fe(II) pulls down His F8 F helix tilts Animation

26. Perutz Mechanism 3. Shift of tertiary structure causes shift of quaternary structure (rotate) a2b1 and a1b2 interface residues realign C-terminal residues break ionic interactions which stabilize T state As R state forms from T state, it adopts ideal conformation for next O2 binding All binding sites are altered, not just the one binding the O2

27. Bohr Effect Conformational change will be accompanied by change in IF�s Change in charge Also, H+ and O2 compete for binding to Hb Relate pH to affinity Bohr effect O2 affinity increases as pH increases Animation (YO2 = q)

28. Regulation of O2 Binding O2 affinity affected by other molecules which can bind to the protein CO2 D-2,3-bisphosphoglycerate (BPG) Binds to T state (central cavity) Does not bind to R state Keeps Hb in deoxy form Decreases O2 affinity Allow ligand to be released Animation

29. Chapter 5 Problems 1-6