Biochemistry 4122004

23 March Lecture

Protein-Protein Interactions II

Today’s topics:

• isothermal titration calorimetry

• phage display

First, to review some of the stuff from last time….

Mutant ovomucoid third domainsbinding to serine proteases

Q: how is this related to drug discovery?

Reference Molecule: Turkey Ovomucoid Third Domain(a Serine Protease Inhibitor)

• All nineteen possibleamino acid substitutionswere made for each ofthe residues shown inblue (total = 190).

• For each inhibitor,binding constantswere measured preciselyfor each of six different serine proteases.

• X-ray structures wereperformed on a subsetof the mutant complexes.

Structure of the complex to TKY-OM3D P1 Pro withStreptomyces griseus Protease B

Bateman et al (2001) J. Mol. Biol. 305, 839.

Qasim et al (2003)Biochemistry 42, 6460.

…and the theorists are now beginning to mine this datato refine their docking programs.

“Bad” prediction “Good” prediction

Lorber et al (2002) Protein Sci. 11, 1393.

If you want to be “hard core” and reallyunderstand 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.

Makarov et al (1998) Biopolymers 45, 469.

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

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

When two proteins form a complex, solvent must be displaced from the interfacial regionsand the conformational freedom (configurationalentropy) of the main chain and side chain atomswill change also.

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

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

Isothermal Titration Calorimetry Yields H of Binding

…and when you have H and G(= -RTlnKa), you can calculate S.

Some examples of experimentally-measured thermodynamic quantities forinteracting 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.

Phage Display

G. P. Smith (1985) “Filamentous fusion phage:novel expression vectors that display cloned

antigens on a virion surface” Science 228, 315.

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

Sidhu et al (2003) ChemBiochem 4, 14.

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

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

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

Other interesting (and related) topics:

• antibody engineering

• yeast “two-hybrid” technology