1. Proteins often consist of multiple domains –Usually different functions (eg. catalysis, regulation, targeting) –Often can be physically separated Non-covalent interactions: 4 structure One polypeptide with multiple ‘independent’ subdomains Protein structures fall into a limited number of categories –Classified according to 2 structure composition  –Conserved motifs seen, with limited variation, in a number of proteins Note: conservation of structure is a great way to determine an evolutionary relationship…better than function or sequence

2. Protein folding is complex –How does a protein “know” how to fold? Completely due to amino acids (some proteins may need assistance from molecular “chaperones”) –Studying protein folding Often through denaturation/renaturation curves: how stable is a protein? How quickly does it (un)fold? –Several imperfect models

3. Reversible binding involving proteins 1.Interactions between proteins 2.Protein/DNA 3.Protein/small molecule ligand

4. Reversible binding involving proteins (Ch. 5) 1.Interactions between proteins –Different from 4° structure Lower affinity (in general) Reversible Potential for numerous partners

5. Hemoglobin Four ‘separate’ polypeptide chains One ‘protein’ Function as a whole Antibody (green)/Antigen (red) Two different proteins Found apart 4° Structure Protein-protein interaction

6. Reversible binding 1. asdf 2.Protein vs. “small” molecule –Protein acts as a carrier for the molecule Hemoglobin/O 2 Metallochaperones –Enzymes Catalyze a reaction involving the substrate 3.Protein-DNA interactions

7. Principles of reversible interactions Affinity of protein for ligand is very specific –eg. high affinity for Mg 2+, low affinity for Zn 2+ –eg. fumarase: distinguishes stereoisomers of tartaric acid Ligand binding site is usually complementary to the ligand BUT ligand binding can cause drastic conformational changes –Induced fit –Conformational changes result in tighter binding but strain both protein and ligand

8. C C INACTIVE PKA C C cAMP binding results in conformational change: regulatory subunits no longer bind catalytic: ACTIVE PKA

9. Principles of reversible interactions Enzymes –Ligands = substrate and product –Induced fit stress can drive catalysis

10. Quantification of protein-ligand interactions (non-catalytic) P + L ↔ PL Reversible: represent as equilibrium K a = [PL] [P][L] Association constant (don’t confuse with K a /pK a ) High K a : [complex] is relatively high ie. protein has a high affinity for the ligand K a * ([L]) = [PL] [P] Amount of complex depends on concentration of free ligand as well as the affinity (K a )

11. Quantification of protein-ligand interactions Work with dissociation constants PL ↔ P + L Equilibrium equation describing dissociation K d = [P][L]Note that K d = 1/K a [PL]

12. Quantification of protein-ligand interactions Assume [L] >> [P] –Few proteins (binding sites), lots of the ligand –ie. conc. of free ligand doesn’t change (much) even if all ligand-binding sites are filled Fraction of ligand binding sites filled (   = [L] [L] + K d

13.  = [L] [L] + K d When [L] = K d,  = 0.5 **When [L] = K d (note: no matter what [P] is (remember assumption, though)), half of the binding sites will be filled Lower K d : need less ligand to fill binding sites Lower K d corresponds to higher affinity/stronger binding

14. % of sites filled vs. [L] Units of K d : concentration (M, mM,  M, etc)

15. Protein x with three different ligands Max binding (  = 1.0, all binding sites filled/saturated) 50% of saturation 1 0.5 0 Fraction of binding sites occupied  Kd1Kd1 Kd2Kd2 Kd3Kd3

17. Case study: oxygen binding in myoglobin and hemoglobin Oxygen is poorly soluble in water (blood) Iron (Fe 2+ )/O 2 complex is soluble –But free iron is toxic Use proteins containing an iron cofactor –Myoglobin –Hemoglobin

18. Iron is part of a heme prosthetic group: permanent association with protein

19. Iron has six coordination sites Four bind heme nitrogens One binds protein histidine “proximal” histidine One can bind O 2

20. Structure of myoglobin Extremely compact ~75%  helix (no  structure) –Eight helical segments –Four terminate in proline Interior: hydrophobic except for two histidines