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A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR were developed to test the ability of wild-type mutant forms of Phosphotriesterase (PTE) enzyme to hydrolyze these agents. Mutant forms of PTE identified with significantly enhanced as well as relaxed or reversed, stereoselectivity were observed to accurately predict a number of variants which exhibited remarkablly improved kinetic constants for the catalytic hydrolysis of the more toxic [S.sub.P] enantiomers.

Four peptides corresponding to alpha-helical regions delimited by residues 63-73 and 97-112 of cytochrome c2 (Rhodospirillum) and residues 24-36 and 45-55 of bovine calcium binding protein are predicted to be alpha-helical by a recently developed method [Rooman, M., Kocher, J.P., & Wodak, S.J. (1991) J. Mol. Biol. 221, 961-979], synthesized by solid phase methods, and purified by HPLC, and their solution conformations are determined by NMR and CD. The observed conformational properties of these peptides in solution confirmed prediction results: in water/TFE (60/40, v/v) at room temperature, these peptides adopt an alpha-helical conformation, as shown by an extended pattern of strong, sequential dNN(i,i + 1) NOE cross-peaks, d alpha N(i,i + 1) NOEs of reduced intensity, several medium-range [d alpha N(i,i + 3), d alpha N(i,i + 4), d alpha beta-(i,i + 3)] NOE connectivities, small 3JH alpha N values, and more upfield alpha-proton chemical shifts. CD studies at different TFE concentrations and at room temperature provide further evidence of the propensity of these peptides to adopt an alpha-helical conformation in solution, as determined by the ellipticity values at 222 nm, and by deconvolution of the CD spectra. According to the method used, helicities in the range 34-50% and 55-75% are found for the 63-73 and 97-112 fragments of cytochrome c2, respectively, and in the range 53-80% and 42-65% for the fragments 24-36 and 45-55 of calcium binding protein in water/TFE (60/40, v/v) at 298 K. In addition, the experiments and predictions agree for those residues that are more flexible. Finally, the relevance of our results for the protein folding pathways is discussed.

The isolated F2-V8 peptide corresponding to the 101 C-terminal residues of Escherichia coli tryptophan synthase beta chains folds into a heat-stable, yet fluctuating, condensed state that contains a lot of secondary structure. However, this state has non-native-like secondary and supersecondary structures [Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.-J., & Goldberg, M. E. (1991) Biochemistry 30, 8067-8074]. To characterize the rate of appearance of this state, stopped-flow studies on the far-ultraviolet circular dichroism (CD) and on the binding of 1-anilino-8-naphthalenesulfonate (ANS) have been conducted during the folding of guanidine-unfolded F2-V8. It was shown that both the CD signal at 222 nm and the ANS binding properties of folded isolated F2-V8 were regained, at 20 degrees C, within the dead time of the stopped-flow apparatus, which was 4 ms. At 12 degrees C, the binding of ANS was also completed within this dead time, but the ellipticity showed some minor later changes. After a rapid overshoot of the CD signal that occurred during the 4-ms dead time, a small readjustment of the ellipticity to the final value occurred more slowly and was completed after about 25 ms. Thus, even at 12 degrees C, the hydrophobic core and most of the secondary structure of folded F2-V8 were formed in less than 4 ms. These observations strongly suggest that the previously described condensed non-native-like state of F2-V8 results from a very rapid, nonspecific, hydrophobic collapse. It is proposed that such a state may be a general early intermediate in protein folding.