Studies of protein dynamics using heteronuclear two-dimensional nuclear magnetic resonance spectroscopy.

The research described herein centers on heteronuclear two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy techniques aimed at characterizing protein dynamics. This has involved addressing both theoretical and experimental aspects of multi-dimensional NMR spectroscopy. The specific experimental emphasis is on the measurement of nuclear spin relaxation parameters of proton-heteronucleus (e.g. $\sp{13}$C$\alpha$-$\sp1$H and amide $\sp{15}$N-$\sp1$H) bond vectors in proteins. The relaxation parameters of these bonds act as local sensors of mobility in the protein, and 2D NMR methods permit relaxation parameters to be measured on a per residue basis. The relaxation parameters give information about the mobility of these bond vectors through their dependence on power spectral density functions. These spectral density functions are essentially the frequency distributions for the rotational dynamics of the bonds with respect to the external magnetic field. The elucidation of these functions is the central goal of NMR spectroscopists focusing on molecular dynamics. There are two main results of the research. First, a strategy has been developed that permits direct experimental access to the aforementioned spectral density functions. This contrasts with previous techniques, which have not provided sufficient information to extract these quantities from experiment. Instead, the use of motional models have been required to cast the relaxation data in a dynamics context, thereby rendering any interpretations dependent on the assumptions of the model. The second main result is application of this strategy to study the backbone dynamics of the $\sp{15}$N-enriched protease inhibitor eglin c, a protein of 70 residues. The spectral density mappings have been complemented by amide hydrogen-deuterium exchange studies. Together, the two approaches reveal greater internal motion for the active site protease binding loop and the first eight residues. Further studies of a non-functional double mutant L45R/D46S have also provided insight into the potential role of dynamics in affecting inhibitory function.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103470/1/9319609.pdfDescription of 9319609.pdf : Restricted to UM users only