1. Protein-Protein Interactions II
Biochemistry 412
2004
23 March Lecture
Protein-Protein Interactions II

2. Today’s topics:
• isothermal titration calorimetry
• phage display

3. Mutant ovomucoid third domains binding to serine proteases
First, to review some of the stuff from last time….
Mutant ovomucoid third domains
binding to serine proteases
Q: how is this related to drug discovery?

4. Reference Molecule: Turkey Ovomucoid Third Domain
(a Serine Protease Inhibitor)
• All nineteen possible
amino acid substitutions
were made for each of
the residues shown in
blue (total = 190).
• For each inhibitor,
binding constants
were measured precisely
for each of six different
serine proteases.
• X-ray structures were
performed on a subset
of the mutant complexes.

5. Structure of the complex to TKY-OM3D P1 Pro with
Streptomyces griseus Protease B
Bateman et al (2001) J. Mol. Biol. 305, 839.

6. Qasim et al (2003)
Biochemistry 42, 6460.

7. …and the theorists are now beginning to mine this data
to refine their docking programs.
“Bad” prediction
“Good” prediction
Lorber et al (2002) Protein Sci. 11, 1393.

8. If you want to be “hard core” and really
understand protein-protein interactions,
you need to know more than just the
free energies of association. You (ultimately)
will need to know something about enthalpies,
entropies, and heat capacities, too.

9. Makarov et al (1998) Biopolymers 45, 469.

10. Makarov et al (2000) Biophys. J. 76, 2966.

11. Makarov et al (2002) Acc. Chem. Res. 35, 376.

12. When two proteins form a complex, solvent
must be displaced from the interfacial regions
and the conformational freedom (configurational
entropy) of the main chain and side chain atoms
will change also.

13. Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3.

14. Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3.

15. Isothermal Titration Calorimetry Yields DH of Binding

16. …and when you have DH and DG
(= -RTlnKa), you can calculate DS.

17. Some examples of experimentally-measured thermodynamic quantities for
interacting proteins, measured using isothermal titration calorimetry:
Note: isothermal titration calorimetry also directly yields n, the stoichiometry of binding.
Weber and Salemme (2003) Curr. Opin. Struct. Biol. 13, 115.

18. Phage Display G. P. Smith (1985) “Filamentous fusion phage:
novel expression vectors that display cloned
antigens on a virion surface” Science 228, 315.

19. Bass et al (1990) Proteins 8, 309
(as referenced in Sidhu (2000)
Curr. Opin. Biotechnol. 11, 610).

20. Sidhu et al (2003) ChemBiochem 4, 14.

21. Sidhu (2001) Biomolec. Eng. 18, 57.

22. Sidhu (2001) Biomolec. Eng. 18, 57.

24. Rodi et al (2002) Curr. Opin. Chem. Biol. 6, 92.

25. Other interesting (and related) topics:
• antibody engineering
• yeast “two-hybrid” technology