NMR studies of folding hierarchies and the role of electrostatics in protein folding
Date of Completion
Understanding the forces involved in protein folding is necessary for the development of reliable modeling methods for protein structure prediction and design and is also important for establishing the mechanisms by which proteins misfold in disease states. To this end we have carried out protein folding studies on a number of systems including globular OB-fold proteins, and the non-globular GCN4 leucine zipper coiled coil. (1) Native state hydrogen exchange NMR experiments were used to compare partially folded intermediates of three OB-fold proteins that lack sequence identity: cold shock protein A (CspA), staphylococcal nuclease (SN), and the anticodon-binding domain of Lys-tRNA synthetase (LysN). The regions of the structures with the largest stability to unfolding were observed to cluster within the structurally conserved OB-fold, suggesting that partially folded intermediates are conserved between the proteins. (2) The leucine zipper region of the yeast transcription activator GCN4 is a well known fibrous protein that spontaneously forms a homodimeric coiled coil. In an effort to understand the roles of surface charge-charge interactions in the stabilization of the coiled coil, we used NMR to obtain pKa values for all acidic and basic residues that ionize in the pH range 1 through 13. The thermodynamic linkage contributions to ΔG unf calculated from the pKa data, suggest that electrostatic interactions contribute significantly to the overall stability of the coiled coil. (3) The proposed 'trigger sequence' of the GCN4 coiled coil system is believed to spontaneously adopt an α-helical structure and to nucleate coiled coil formation. The structure of the C-terminal trigger peptide of GCN4 was determined by NMR, and the polar contacts that stabilize structure in the peptide were identified. A pH dependent ion pair, formed between Arg25 and Glu22, was found to be critical for structure. (4) Examination of the denatured state of the GCN4 coiled coil system using residual dipolar coupling (RDC) and other NMR techniques revealed that some residual α-helix structure persists even under strongly denaturing conditions (6 M urea). These observations highlight the hierarchical ordering of protein structure and suggest an important role for electrostatic interactions for the formation and maintenance of structure. ^
Matousek, William Michael, "NMR studies of folding hierarchies and the role of electrostatics in protein folding" (2007). Doctoral Dissertations. AAI3269492.