UCSF

Proteins are complex macromolecules whose structure informs their function and regulation in difficult to predict ways. Understanding their shape, dynamics, and regulation all pose major challenges in terms of collecting, analyzing, and interpreting data. In my dissertation I describe two contributions to data analysis for determining the structure and dynamics of proteins using novel approaches, as well as experimental work querying the function of an enzyme with a particularly recalcitrant substrate.

In the first chapter of this dissertation, I develop a tool, EMRinger, for the emerging field of high resolution electron microscopy that takes advantage of prior physical information about model geometry to more effectively determine if the model is built correctly into the map. This work adapted the tool Ringer, which had been previously developed in the Alber lab, in order to identify the dihedral angle for side chains with the greatest density, and confirm that the distribution of those peak positions does not violate the constraints of side chain dihedral angles to rotameric positions. This approach allows for orthogonal validation of backbone position in density (using the side chain density as a “lever”), which generally improves with refinement and is among the most sensitive model-in-map validation tools available for high resolution electron microscopy.

In the second chapter, I present progress on applying temperature jumps to folded proteins to quantify kinetics of the intrinsic motions in proteins that impact their function and regulation. Using a pulsed infrared laser, we raise the temperature of a protein solution in nanoseconds, and follow the progression of the structure of the protein using solution x-ray scattering. As the protein changes temperature, the conformational equilibrium of the protein shifts as higher energy states become more accessible. By following this progress over the nanoseconds, microseconds and milliseconds following the heating pulse, we are able to reconstruct the relaxation of the protein into its new comformational equilibrium. With this information, we can gain kinetic information about the conformational landscapes of our sample, and using mutations we correlate the rates we observe with existing structural models of dynamics that have been characterized by x-ray crystallography.

In the third chapter, I investigate the mechanics of Acidic Mammalian Chitinase, which has the role of breaking down the recalcitrant polysaccharide chitin in the stomach and lungs of mammals. Mutations to acidic mammalian chitinase have previously been identified that lead to either protection against allergic asthma, and previous work has determined that these mutations lead to an increase in the activity of the enzyme. In order to better determine how these mutations affect activity, I developed new methods to assay chitinase activity. I use these methods to characterize the effects of the asthma-associated mutations, as well as investigating the role of the individual domains of acidic mammalian chitinase in degrading crystalline chitin and the differences in behavior of acidic mammalian chitinase and the other chitinase expressed in mammals, chitotriosidase. Additionally, I attempt to engineer hyperactive chitinases, and show that direct evolution based on screening with traditional fluorogenic oligomer substrates does not necessarily lead effectively to enzymes which are hyperactive against complex substrate, emphasizing the need for sensitive and high throughput methods to quantify degradation of crystalline chitin.