Microscopic stability of cold shock protein A examined by NMR native statehydrogen exchange as a function of urea and trimethylamine N-oxide

Native state hydrogen exchange of cold shock protein A (CspA) has been char acterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of b eta-sheet. Strands beta 1-beta 4 have strongly protected amide protons that , based on experiments as a function of urea, exchange through a simple all -or-none global unfolding mechanism. By contrast, the protection of amide p rotons from strand beta 5 is too weak to measure in water. Strand beta 5 is hydrogen bonded to strands beta 3 and beta 4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact den sity in the native structure, accurately predict the strong protection obse rved in strands beta 1-beta 4 but fail to account for the weak protection i n strand beta 5. The most conspicuous feature of strand beta 5 is its low s equence hydrophobicity. In the presence of TMAO, there is an increase in th e protection of strands beta 1-beta 4, and protection extends to amide prot ons in more hydrophilic segments of the protein, including strand beta 5 an d the loops connecting the beta-strands. TMAO stabilizes proteins by raisin g the free energy of the denatured state, due to highly unfavorable interac tions between TMAO and the exposed peptide backbone. As such, the stabilizi ng effects of TMAO are expected to be relatively independent of sequence hy drophobicity. The present results suggest that the magnitude of solvent exc hange protection depends more on solvent accessibility in the ensemble of e xchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.