Andrei T. Alexandrescu

Hydrogen bonding in cold-shock protein A of Escherichia coli has been investigated using long-range HNCO spectroscopy. Nearly half of the amide protons involved in hydrogen bonds in solution show no measurable protection from exchange in water, cautioning against a direct correspondence between hydrogen bonding and hydrogen exchange protection. The N to O atom distance across a hydrogen bond, R_NO, is related to the size of the ^3hJ_NC'trans hydrogen bond coupling constant and the amide proton chemical shift. Both NMR parameters show poorer agreement with the 2.0-Å resolution X-ray structure of the cold-shock protein studied by NMR than with a 1.2-Å resolution X-ray structure of a homologous cold-shock protein from the thermophile B. caldolyticus. The influence of crystallographic resolution on comparisons of hydrogen bond lengths was further investigated using a database of 33 X-ray structures of ribonuclease A. For highly similar structures, both hydrogen bond R_NO distance and Cα coordinate root mean square deviations (RMSD) show systematic increases as the resolution of the X-ray structure used for comparison decreases. As structures diverge, the effects of coordinate errors on R_NO distance and Cα coordinate root mean square deviations become progressively smaller. The results of this study are discussed with regard to the influence of data precision on establishing structure similarity relationships between proteins.

Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) ^h3J_NC′ scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The ^h3J_NC′ couplings give direct evidence for the closing of individual backbone N-H•••O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no ^h3J_NC′ correlations could be detected without TFE, α-helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that α-helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed α-helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the ^h3J_NC′ constants represent ensemble averages and the dependence of ^h3J_NC′ on H-bond lengths is very steep, the size of the individual ^h3J_NC′ coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings.

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N‐oxide (TMAO). The structure of CspA has five strands of β‐sheet. Strands β1‐β4 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 protons from strand β5 is too weak to measure in water. Strand β5 is hydrogen bonded to strands β3 and β4, 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 density in the native structure, accurately predict the strong protection observed in strands β1‐β4 but fail to account for the weak protection in strand β5. The most conspicuous feature of strand β5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands b̊1‐β4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand β5 and the loops connecting the β‐strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen‐bonding interactions in the native structure.