Protein crystallography: development of new methods, and application to the study of pathogenesis.

The proposed research is in the area of protein crystallography, involving both the development of new methods, and applications to the study of proteins relevant to disease. Crystallographic methods in our new computer program Phaser are based on the principle of maximum likelihood, which requires an understanding of the statistical relationships among experimental observations. We will build on success in the area of molecular replacement to solve structures with less prior knowledge, usin g either smaller fragments or ab initio models. We will complete the implementation of experimental phasing by multiple-wavelength isomorphous replacement and two-wavelength anomalous diffraction, and we will explore other applications, for instance to phasing in the presence of radiation damage. In our structural research, we will complete our study of the enzymatic mechanism of pertussis toxin by determining structures representing the Michaelis complex. We will study the mechanism by whi ch the hormone-binding globulins carry out a targeted release of bound hormone, and will attempt to exploit that understanding to design variants that release selected drugs at selected target sites. Finally, we will begin a new study on enzymes mutated in lysosomal storage diseases, with the eventual goal of contributing to improvements in enzyme replacement therapy.