Chapter 5.1: Protein Function - Reversible Binding of Protein to a Ligand

1. Chapter 5.1: Protein Function - Reversible Binding of Protein to a Ligand CHEM 7784 Biochemistry Professor Bensley

2. CHAPTER 5.1Reversible Binding of Protein to a Ligand • Reversible binding of ligands • Structure of myoglobin and hemoglobin • Origin of cooperativity in hemoglobin Today’s Objectives - To learn and understand:

3. Functions of Globular Proteins • Storage of ions and molecules • myoglobin, ferritin • Transport of ions and molecules • hemoglobin, serotonin transporter • Defense against pathogens • antibodies, cytokines • Muscle contraction • actin, myosin • Biological catalysis • chymotrypsin, lysozyme

5. Binding: Quantitative Description • Consider a process in which a ligand (L) binds reversibly to a site in the protein (P) • The equilibrium composition is characterized by the equilibrium constant Ka ka + L PL P kd

6. Binding: Analysis in Terms of the Bound Fraction • In practice, we can often determine the fraction of occupied binding sites • Substituting [PL] with Ka[L][P], we’ll eliminate [PL] • Eliminating [P] and rearranging gives the result in terms of equilibrium association constant: • In terms of the more commonly used equilibrium dissociation constant:

7. Binding: Graphical Analysis • The fraction of bound sites depends on the free ligand concentration and Kd • In a typical experiment, ligand concentration is the known independent variable • Kd can be determined graphically or via least-squares regression [L]  [L]total

9. Specificity: Lock-and-Key Model • “Lock and Key” model by Emil Fischer (1894) assumes that complementary surfaces are preformed. +

10. Specificity: Induced Fit • Conformational changes may occur upon ligand binding (Daniel Koshland in 1958). • This adaptation is called the inducedfit. • Induced fit allows for tighter binding of the ligand • Induced fit can increase the affinity of the protein for a second ligand • Both the ligand and the protein can change their conformations +

11. Myoglobin/Hemoglobin • First protein structures determined • Oxygen carriers • Hemoglobin: transportsO2 from lungs to tissues • Myoglobin: O2storage protein

12. Mb and Hb Subunits Structurally Similar • 8 alpha-helices • Contain heme group • Mb monomeric protein • Hbheterotetramer (α2β2) myoglobin hemoglobin

13. Heme Group

14. Structure of Myoglobin

16. Hemoglobin

17. Oxygen Binding Curves • Mb has hyberbolic O2 binding curve • Mb binds O2 tightly. Releases at very low pO2 • Hb has sigmoidal O2 binding curve • Hb high affinity for O2 at high pO2 (lungs) • Hb low affinity for O2 at low pO2 (tissues)

18. Oxygen Binding Curve

19. Oxygen Binding Curve

20. O2 Binding to Hb shows Positive Cooperativity • Hb binds four O2 molecules • O2 affinity increases as each O2 molecule binds • Increased affinity due to conformation change • Deoxygenated form = T (tense) form = low affinity • Oxygenated form = R (relaxed) form = high affinity

21. O2 Binding to Hb shows Positive Cooperativity

22. Conformational Change is Triggered by Oxygen Binding

23. Video on Hemoglobin

25. Allosteric Interactions • Allosteric interaction occurs when specific molecules bind a protein and modulate activity • Allosteric modulators or allosteric effectors • Bind reversibly to site separate from functional binding or active site • Modulation of activity occurs through change in protein conformation • 2,3 bisphosphoglycerate (BPG), CO2 and protons are allosteric effectors of Hb binding of O2

26. Regulation of O2 Binding by 2,3-Bisphospho-glycerate