Assistant Professor (biochemistry). Ph.D., California Institute of Technology, 1994; National Science Foundation Postdoctoral Fellow, Scripps Research Institute, 1994-1996.
Biomolecular systems display vast complexity, diversity and specificity, yet structure and recognition are controlled by the combination of simple, fundamental molecular interactions. These interactions include hydrogen bonding, hydrophobic interactions, ionic bonding, and steric interactions. Although considerable progress has been made in the field of molecular recognition in biological systems, a more detailed quantitative description of the thermodynamic and kinetic factors governing these interactions is essential for an in-depth understanding of many biological processes, which can lead to improvements in the efficacy of bioengineering and drug design. A diverse and complementary set of techniques is used in our laboratory to study the thermodynamic and kinetic control of molecular recognition in a series of biological systems. This is achieved by the integration of powerful physical techniques, including nuclear magnetic resonance (NMR), time-resolved and steady-state fluorescence and circular dichroism spectroscopies, with advanced methodologies for the preparation of biomolecules aimed at determining the specific chemical basis for molecular recognition and protein folding. The foremost difficulty in studies of molecular recognition is finding biologically relevant systems of suitable size, stability and specificity that have the capacity to yield detailed physical information. Our initial efforts will focus on the interactions between peptides as well as interactions within proteins. Non-covalent complexes of fragments of proteins are prepared and characterized as models for both protein/protein molecular recognition and the interactions governing correct protein folding. Our initial approach focuses on complexes comprising fragments of proteins that can be reconstituted into structurally intact proteins from their smaller component fragments, including SH2 domains and a/b barrel proteins. These non-covalently linked complexes surprisingly exhibit residual activity of the native protein. We are interested in determining the structural requirements for this productive complex formation.
In a subsequent step, structurally intact proteins are fully reconstituted from complexes of their component fragments. As peptides of up to sixty amino acids are currently amenable to direct chemical synthesis, this semisynthetic methodology can be applied to readily incorporate both naturally coded and non-coded amino acids directly into the polypeptide backbone at explicitly defined positions. This approach allows us to introduce novel amino acids, such as D-amino acids and unusual sidechain structures, as well as NMR-active isotope labels, at specific sites in the proteins and peptides. Site-specific incorporation of isotope labels at defined positions in the polypeptide chain is being used to obtain time-resolved structural information on proteins as they fold into their native structure by NMR techniques. The high-resolution physical characterization of these systems will be complemented by rational design and modeling efforts, eventually accommodating the introduction of functional sites and redox centers directly into proteins.
NMR spectroscopy is used not only to obtain high-resolution structural information pertaining to recognition features in the proteins and complexes studied, but also to characterize protein-bound water molecules and to investigate the role of dynamics and exchange processes in mediating catalysis and recognition. While it is intuitively evident that the dynamical properties of a biomolecule will affect its recognition activity, little evidence is available detailing the role of these processes occurring on timescales ranging from picoseconds to days in mediating catalysis and recognition. The uniform and site-specific incorporation of isotope labels into proteins will facilitate the assignment and structural characterization of specific recognition interactions and also provide probes of dynamical parameters critical to complex formation and protein folding.
Meier, R. van Eldik, I-J. Chang, G.A. Mines, D.S. Wuttke, J.R. Winkler and H.B. Gray. "Pressure Effects on the Rates of Intramolecular Electron Transfer in Ruthenium-Modified Cytochrome c. Role of the Interventng Medium in Tuning Distant Fe2+:Ru3+ Electronic Couplings," J. Am. Chem. Soc. 116, 1577-1578 (1994).
D.S. Wuttke, H.B. Gray, S.L. Fisher and B. Imperiali. "Semisynthesis of Bipyridyl-Alanine Cytochrome c Mutants: Novel Proteins with Enhanced Electron-Transfer Properties," J. Am. Chem. Soc. 115, 8455-8456 (1993).
D.S. Wuttke and H.B. Gray. "Protein Engineering as a Tool for Understanding Electron Transfer," Curr. Opin. Struct. Biol. 3, 555-563 (1993).
D.S. Wuttke, M.J. Bjerrum, J.R. Winkler and H.B. Gray.
"Electron-Tunneling Pathways in Cytochrome c," Science
256, 1007-1008 (1992)