Miniproteins are adequate models to study various protein-structure modifying effects such as temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W. et al. (2002) Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. Here we report a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6 and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions, selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. These observations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.
An increasing number of diseases, including Alzheimer's, have been found to be a result of the formation of amyloid aggregates that are practically independent of the original primary sequence of the protein(s). (Eakin, C. M.; Berman, A. J.; Miranker, A. D. Nat. Struct. Mol. Biol. 2006, 13, 202-208.) Consequently, the driving force of the transformation from original to disordered amyloid fold is expected to lie in the protein backbone, which is common to all proteins. (Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.; Grothe, R.; Eisenberg, D. Nature 2005, 435, 773-778. Wright, C. F.; Teichmann, S. A.; Clarke, J.; Dobson, C. M. Nature 2005, 438, 878-881.) However, the exact explanation for the existence of such a "dead-end" structure is still unknown. Using systematic first principle calculations on carefully selected but large enough systems modeling the protein backbone we show that the beta-pleated sheet structure, the building block of amyloid fibers, is the thermodynamically most stable supramolecular arrangement of all the possible peptide dimers and oligomers both in vacuum and in aqueous environments. Even in a crystalline state (periodical, tight peptide attechment), the beta-pleated sheet assembly remains the most stable superstructure. The present theoretical study provides a quantum-level explanation for why proteins can take the amyloid state when local structural preferences jeopardize the functional native global fold and why it is a beta-pleated sheetlike structure they prefer.
Proton affinity and pK a values of N-formyl-l-histidinamide are found to vary as a function of its backbone and/or side-chain orientation. Proton affinities between the cationic and neutral forms of structurally similar conformers are between −246 and −230 kcal mol-1, while pK a values associated with the same conformers are between 6 and 8. For the neutral-to-anion transition, the following ranges were computed −342 > PA > −350 kcal mol-1 and 18 < pK a < 22. The protonation state of histidines on the surface of a protein depends primarily on the pH. Due to protonation or deprotonation, the side-chain and/or backbone orientation of these histidine residues may undergo considerable changes. Examples are presented and confirmed by ab initio calculations, where proteins were crystallized under various pH conditions, resulting in the same histidine residue to adopt different conformations. Furthermore, a hypothesis is given for a protonation-induced conformational modification of the histidine residue in the catalytic triad of chymotrypsin during catalysis, which lowers the pK a value of the catalytic histidine by 1.2 units. Both the experimental and theoretical results support that proton affinity as well as that pK a values of histidine residues are strongly conformationally dependent.
Abstract:The present study focuses on important questions associated with modeling of peptide and protein stability.Computing at different levels of theory (RHF, B3LYP) for a representative ensemble of conformers of di-and tripeptides of alanine, we found that the Gibbs Free Energy values correlate significantly with the total electronic energy of the molecules (0.922 Յ R 2 ). For noncovalently attached but interacting peptide subunits, such as [For-NH 2 ] 2 or [For-L-Ala-NH 2 ] 2 , we have found, as expected, that the basis set superimposition error (BSSE) is large in magnitude for small basis set but significantly smaller when larger basis sets [e.g., B3LYP/6-311ϩϩG(d,p)] are used. Stability of the two hydrogen bonds of antiparallel -pleated sheets were quantitatively determined as a function of the molecular structure, S10 and S14, computed as 4.0 Ϯ 0.5 and 8.1 Ϯ 1.1 kcal/mol, respectively. Finally, a suitable thermoneutral isodesmic reaction was introduced to scale both covalently and noncovalently attached peptide units onto a common stability scale. We found that a suitable isodesmic reaction can result in the total electronic energy as well as the Gibbs free energy of a molecule, from its "noninteracting" fragments, as accurate as a few tenths of a kcal per mol. The latter observation seems to hold for peptides regardless of their length (1 Յ n Յ 8) or the level of theory applied.
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