Title

NMR studies of structural motifs involved in protein folding and ligand binding

Date of Completion

January 2008

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The agrin G3 domain plays a critical role in the development and maintenance of the neuromuscular junction by binding to α-dystroglycan and initiating acetylcholine receptor clustering on the post-synaptic membranes of myotubes. Using NMR spectroscopy, we show that both the active B8 and inactive BO isoforms bind to sialic acid, heparin, and heparan sulfate, and have mapped the binding sites. Dissociation constants for these ligands were determined using isothermal titration calorimetry, and pulse field gradient diffusion NMR was used to determine the stoichiometry of heparin binding, which was found to be two protein molecules per heparin. In another series of investigations, residual dipolar couplings (RDCs) were used to probe residual structure in the denatured states of OB-fold proteins. It was shown that folded forms of staphylococcal nuclease (SN) have greater RDC differences than the unfolded forms. The invariance of RDCs in denatured forms of SN to changes in denaturant concentration or amino acid sequence had previously been attributed to the robustness of long-range structure in the denatured state. We show, however, that the increased RDC differences in the folded states are a consequence of the close packing of the polypeptide chain under native conditions. This is more consistent with a disruption of cooperative structure in the folded states than with the retention of a unique long-range folding topology in the denatured states. RDCs were used to locate possible residual secondary structure in β-sheet segments of the denatured form of the OB-fold LysN and CspA. ^