Structural characterization of the unfolded states of an SH3 domain and a partially folded fragment of staphylococcal nuclease by NMR, methods and applications

grantor: University of TorontoThe isolated N-terminal SH3 domain of the Drosophila signaling protein drk exists in an equilibrium between folded and unfolded states under conditions close to physiological. This system provides an interesting model for comparison of preferential conformations of unfolded states in near physiological conditions with unfolded states under denaturing conditions and also for studying the effects of binding of peptides or proteins on the equilibrium between folded and unfolded states. This doctoral work involved structural and dynamic characterization of folded and unfolded states of this SH3 domain, under both folding and strong denaturing conditions by multi-nuclear, multi-dimensional NMR. The effect of binding a proline-rich peptide derived from the protein Sos, the biological target of the drkN SH3 domain, on the equilibrium between the folded and unfolded states of this domain has been investigated. The stabilization of the folded structure of the domain upon binding provides an example of coupling of binding to protein folding or stabilization. Our data for both unfolded states demonstrate that similar turn-like structures throughout the protein may function to bring parts of the polypeptide chain that are distant in primary sequence into close spatial proximity and initiate protein folding through multiple initiation sites. In spite of the overall structural similarity of both unfolded states, marked differences in detailed structural features between the two unfolded states argue that caution is needed in interpretation of results from structural characterization of protein conformational states generated using denaturing conditions. Structural determination of the folded state stabilized in 400 mM $\rm Na\sb2SO\sb4$ yields a structure very similar to previously published SH3 domain structures. New triple-resonance NOESY-based NMR experiments with improved resolution for application to structural characterization of unfolded or partially folded proteins have been developed in this thesis work. The experiments can also be applied to folded protein states. These novel methods greatly facilitate unambiguous assignment of NOEs in partially folded or unfolded protein systems which would be impossible to identify from conventional experiments due to severe resonance overlap. Results from application of these methods to a partially folded large fragment of staphylococcal nuclease ($\Delta$131$\Delta)$ and the drkN SH3 domain are used to obtain general NOE patterns for unfolded or partially folded proteins. Especially worth noting are our observations of NOEs which are not normally observed in folded, native proteins. These results suggest that unfolded or partially folded proteins may allow sampling of non-prolyl cis peptide bond configurations. These NMR methods developed in our laboratories should provide an experimental basis for more comprehensive analysis of other unfolded or partially folded proteins.Ph.D