Single-site mutation and secondary structure stability: an isodesmic reaction approach. The case of unnatural amino acid mutagenesis Ala→Lac

A method is described to evaluate backbone interactions in proteins via computational unnatural amino acid mutagenesis. Several N-acetyl polyalanyl amides (AcA nNH 2) were optimized in the representative helical (3 10-, 4 13-, and a "hybrid" κ-helix, n = 7, 9, 10, 14) and hairpin (two- and three-stranded antiparallel β-sheets with type I turns βααε, n =6, 9, 10) conformations, and extended conformers of N-acetyl polyalanyl methylamides (n = 2, 3) were used to derive multistranded β-sheet fragments. Subsequently, each residue of every model structure was substituted, one at a time, with L-lactic acid. The resulting mutant structures were again optimized, and group-transfer energies ΔE GT were obtained as heats of the isodesmic reactions: AcA nNHR + AcOMe → AcA xLacA yNHR + AcNHMe (R = H, C H 3). These group-transfer energies correlate with the degree of charge polarization of the substituted peptide linkages as measured by the difference Δe in H and O Mulliken populations in HN-C=O and with the H-bond distances in the "wild-type" structures. A good correlation obtains for the HF/3-21G and B3LYP/6-31G* group-transfer energies. The destabilization effects are interpreted in terms of loss of interstrand and intrastrand H-bonds, decrease in Lewis basicity of the C-O group, and O⋯O repulsion. On the basis of several comparisons of Ala → Lac ΔE GT's with heats of the NH → CH 2 substitutions, the latter contribution is estimated (B3LYP/6-31G*) to range between 1.5 and 2.4 kcal mol -1, a figure close to the recent experimental ΔδG° value of 2.6 kcal mol -1 (McComas, C. C.; Crowley, B. M.; Boger, D. L. J. Am. Chem. Soc. 2003, 125, 9314). The partitioning yields the following maximum values of the electronic association energy of H-bonds in the examined sample of model structures (B3LYP/6-31G* estimates): 3 10-helix D e = -1.7 kcal mol -1, α-helix D e = -3.8 kcal mol -1 β-sheet D e = -6.1 kcal mol -1. The premise of experimental evaluations of the backbone-backbone H-bonding that Ala → Lac substitution in proteins is isosteric (e.g., Koh, J. T.; Cornish, V. W.; Schultz, P. G. Biochemistry 1997, 36, 11314) is often but not always corroborated. Examination of the integrity of H-bonding pattern and ψ i, ψ i distribution identified several mutants with significant distortions of the "wild-type" structure resulting inter alia from the transitions between i, i + 3 and i, i + 4 H-bonding in helices, observed previously in the crystallographic studies of depsipeptides, (Ohyama, T.; Oku, H.; Hiroki, A.; Maekawa, Y.; Yoshida, M.; Katakai, R. Biopolymers 2000, 54, 875; Karle, I. L.; Das, C.; Balaram, P. Biopolymers 2001, 59, 276). Thus, the isodesmic reaction approach provides a simple way to gauge how conformation of the polypeptide chain and dimensions of the H-bonding network affect the strength of backbone-backbone C=O⋯HN bonds. The results indicate that the stabilization provided by such interactions increases on going from 3 10-helix to α-helix to β-sheet.