Biochemistry 412 2004 23 March Lecture Protein-Protein Interactions II
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Transcript of Biochemistry 412 2004 23 March Lecture Protein-Protein Interactions II
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Biochemistry 4122004
23 March Lecture
Protein-Protein Interactions II
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Today’s topics:
• isothermal titration calorimetry
• phage display
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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?
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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.
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Structure of the complex to TKY-OM3D P1 Pro withStreptomyces griseus Protease B
Bateman et al (2001) J. Mol. Biol. 305, 839.
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Qasim et al (2003)Biochemistry 42, 6460.
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…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.
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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.
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Makarov et al (1998) Biopolymers 45, 469.
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Makarov et al (2000) Biophys. J. 76, 2966.
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Makarov et al (2002) Acc. Chem. Res. 35, 376.
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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.
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Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3.
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Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3.
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Isothermal Titration Calorimetry Yields H of Binding
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…and when you have H and G(= -RTlnKa), you can calculate S.
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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.
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Phage Display
G. P. Smith (1985) “Filamentous fusion phage:novel expression vectors that display cloned
antigens on a virion surface” Science 228, 315.
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Bass et al (1990) Proteins 8, 309 (as referenced in Sidhu (2000)Curr. Opin. Biotechnol. 11, 610).
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Sidhu et al (2003) ChemBiochem 4, 14.
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Sidhu (2001) Biomolec. Eng. 18, 57.
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Sidhu (2001) Biomolec. Eng. 18, 57.
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Rodi et al (2002) Curr. Opin. Chem. Biol. 6, 92.
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Other interesting (and related) topics:
• antibody engineering
• yeast “two-hybrid” technology
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