1. Molecular modeling of protein-ligand interactions: Detailed simulations of a biotin-streptavidin complex Prof. Terry P. Lybrand Vanderbilt University Center for Structural Biology & Departments of Chemistry and Pharmacology T. P. Lybrand SURA 2003

2. Streptavidin Protein isolated from Streptomyces species Binds biotin with extremely high affinity Tetramer - ~500 amino acids One biotin binding site per monomer T. P. Lybrand SURA 2003

3. Biotin One of the B-complex vitamins Necessary for CO 2 addition reactions in biosynthesis T. P. Lybrand SURA 2003

4. General Strategy Protein engineering Microcalorimetry X-ray crystallography Molecular simulation T. P. Lybrand SURA 2003

9. Molecular Dynamics AMBER version for distributed parallel computation AMBER version for distributed parallel computation (Swanson & Lybrand, J. Comput. Chem. 16:1131-40 (1995) (Swanson & Lybrand, J. Comput. Chem. 16:1131-40 (1995) T. P. Lybrand SURA 2003

10. Potential Function T. P. Lybrand SURA 2003

11. Potential of Mean Force r c is a reaction coordinate that defines a physical path for the process of interest T. P. Lybrand SURA 2003

12. Calculated vs. Measured Free Energies (Kcal/mol) PMFExpt.*  G˚17.0 ± 3.018.3 ± 1.0 (  H˚ = 24.5 ± 0.5; T  S = 6.2 ± 0.5)  G ‡ 22.0 ± 3.024.4 ± 2.4 (  H ‡ = 32.0 ± 2.1; T  S ‡ = 7.6 ± 2.1) *Chilkoti & Stayton, J. Am. Chem. Soc. 117:10622-8 (1995) T. P. Lybrand SURA 2003

14. Freitag, Chu, Penzotti, Klumb, To, Le Trong, Lybrand, Stenkamp, & Stayton Proc. Natl. Acad. Sci. USA 96:8384-8389 (1999) T. P. Lybrand SURA 2003

15. Hyre, Amon, Penzotti, LeTrong, Stenkamp, Lybrand, & Stayton Nature Struct. Biol. 9:582-585 (2002) T. P. Lybrand SURA 2003

20. Conclusions Detailed simulations can reproduce quantitatively the experimental thermodynamic results Contributions of specific protein residues to net thermodynamic changes can be characterized Simulations can provide detailed insight into dynamics of complex formation and dissociation Specific water molecules may play a crucial role in complex formation and in stabilization of intermediates on the dissociation reaction coordinate Streptavidin appears to have evolved a “back door” water channel, possibly to facilitate solvent evacuation of the binding site during complex formation T. P. Lybrand SURA 2003

21. Dr. Julie E. Penzotti Dr. Lynn Amon Dr. David Hyre Dr. Stephanie Freitag Dr. Lisa Klumb Dr. Richard To Dr. Ashok Chilkoti Vano Chu Isolde LeTrong Prof. Patrick Stayton Prof. Ronald Stenkamp NIH NS33290 NIH DK49655 NIH AI44609 NIH GM56307 NSF MCB9405405 ZymoGenetics T. P. Lybrand SURA 2003