Measurements of Protein Dynamics to Elucidate Protein and Enzyme Function.

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Proteins are molecular machines that carry out most of the work of a living cell. For example, they are responsible for synthesizing and repairing nearly all of the molecules that make up a cell. Proteins carry out their biological activities by changing their shapes in response to interactions with other molecules. This has been known for a long time but the detailed mechanisms of how proteins change their shapes have been difficult to determine. That's because measuring the details requires tracking the position of each atom in a protein on a timescale of billionths and millionths of a second. This field of study is called “protein dynamics” and it promises to reveal the deepest understanding of the mechanisms by which proteins function. Over the next 5 year cycle of my NSERC Discovery program, my students and I will apply the techniques of NMR spectroscopy, mass spectrometry, hydrogen exchange chemistry, X-ray diffraction and molecular dynamics simulations to measure the dynamics of three proteins over a wide range of timescales in order to connect the atomic dynamics to the emergent biological activity of each of the proteins. Advances in the field of protein dynamics will have a profound effect on our understanding of life and will have practical applications in biotechnology, industrial catalysis and drug design. The research will be conducted by graduate and undergraduate students who will be trained in the most advanced methods in the theory and practice of protein dynamics. ******The three proteins to be studied span the landscape of protein structural types including a highly flexible and disordered protein, a much more rigid protein and one that is intermediate between the two. The Transactivator of transcription from the Human Immunodeficiency Virus is highly flexible. We will measure its dynamics by NMR spectroscopy and hydrogen exchange chemistry and we will simulate its dynamics using computers. This work will contribute to a deeper understanding of how binding induces folding and the expression of function in this protein that is essential to viral replication. The glycerol facilitator is a relatively rigid integral membrane protein that permits the passive diffusion of glycerol across the bacterial inner membrane. We propose to measure protein dynamics on this protein in a membrane by measuring backbone amide hydrogen exchange rates with the use of mass spectrometry. These measurements will help delineate the role of dynamics in the transport of glycerol. The third protein we propose to investigate is the viral Ovarian Tumour domain deubiquitinase. It is an enzyme, a protein that catalyzes an important metabolic reaction involving ubiquitin. To probe the role of enzyme dynamics in the catalytic mechanism of this enzyme we will measure NMR relaxation rates over a range of temperatures in the presence and absence of binding partners to construct a comprehensive profile of the enzyme's dynamics. **