Distributions of structural features contributing to thermostability in mesophilic and thermophilic alpha/beta barrel glycosyl hydrolases.

Analysis of the structural basis for thermostability in proteins has come mainly from pairwise comparisons of mesophilic and thermophilic structures and has often yielded conflicting results. Interpretation of these results would be enhanced by knowing the normal range of features found for mesophilic proteins. In order to provide the average and distribution values of structural features among similar mesophilic proteins, we compared the amino acid composition, solvent accessible surface area, hydrogen bonds, number of ion pairs, and thermal factors of 22 structures of alpha/beta barrel glycosyl hydrolases. These distributions are then compared to values from seven alpha/beta barrel glycosyl hydrolases from thermophilic organisms. We find that the distribution of each structural feature is broad within the mesophilic proteins and illustrates the difficulty of making pairwise comparisons of mesophiles to thermophiles where differences for individual proteins may be within the normal range for the group. In comparing mesophiles to thermophiles as a group, we find that thermophilic structures have fewer glycines in a particular region of the structure and higher thermal factors at room temperature. These results suggest the basis for thermostability may be related to protein motion rather than to static features of protein structure.

Approaches for deciphering the structural basis of low temperature enzyme activity.

An increasing number of enzymes active at low temperature are being studied to help determine the structural features important for cold-activity. This review examines the diversity of prokaryotic cold-active enzymes and the features proposed to account for low temperature activity. We then consider the difficulty of identifying the key structural features needed for cold-activity and the need to compare enzymes having different temperature optima from phylogenetically related organisms to determine features responsible for low temperature activity. In addition to studying naturally occurring enzymes, directed evolution experiments are discussed as methods for examining the proposed mechanisms influencing the thermal dependence of activity.

Inductive effects on the structure of proline residues.

4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)- fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C gamma-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1,2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.