Abstract: Objective To elucidate the regulatory principles of how single-point amino acid mutations modulate polypeptide thermal stability, and reveal the holistic regulatory effects mediated by the local properties of side chains. Methods The keratin 14-derived peptide QNL system, comprising the wild-type QNL-R and three mutants (QNL-H, QNL-G, and QNL-S), was synthesized. AlphaFold3 was employed to predict the structural impacts of the single-point mutations. Conformational change of the polypeptide during the heating process was monitored by temperature-dependent intrinsic fluorescence spectroscopy. Based on a two-state transition model, Gibbs free energy (ΔG)and crossover temperature were calculated to quantitatively assess the effect of mutations on thermal stability. Results AlphaFold3 predictions showed a high structural similarity with the wild-type and mutant peptides(backbone RMSD=0.22 Å). However, variable-temperature fluorescence experiments revealed significant physicochemical discrepancies: at 25 ℃, the single-point mutations significantly altered the intrinsic fluorescence emission intensity by remodeling the local chemical microenvironment(P＜0.05). During the heating process, although all four peptides exhibited similar conformational evolution trends(decreased fluorescence intensity at 303 nm indicating unfolding, and increased fluorescence intensity at 391 nm indicating thermal aggregation), thermodynamic analysis demonstrated that the mutations significantly altered the thermal stability of the peptides which lead to systematic shift in the crossing-point temperatures as follows: QNL-G(55.9 ℃)＞QNL-H(54.7 ℃)＞QNL-S(54.2 ℃)＞QNL-R(52.2 ℃). Conclusions Single-point amino acid mutations alter the physicochemical properties of residue side chains and have significant impact on the overall conformational thermal stability of the peptides in a non-additive manner. This finding shows the limitation of theoretical predictions of protein structure on static conformations and the principle of energy minimization.

Key words: keratin-derived peptide, single-point mutation, thermal stability, aggregation, intrinsic fluorescence