Effects of Amino Acid Point Mutations on the Local Hydrophobicity, Structural Stability, and Conformational Flexibility of P-Glycoprotein.

P-glycoprotein (P-gp, ABCB1) plays a central role in multidrug resistance and drug pharmacokinetics. In this study, an integrated computational analysis of point amino acid substitutions across the P-gp sequence was performed to evaluate their predicted pathogenicity and structural impact. Variant classification using AlphaMissense and PolyPhen-2 revealed concordant predictions for a substantial subset of amino acid substitutions, with 21 variants consistently classified as benign and 13 as pathogenic, while discrepancies for other variants reflected methodological differences between the tools. Notably, substitutions located in transmembrane domains were more frequently predicted to be deleterious compared with those in cytoplasmic or extracellular regions, consistent with the structural and functional constraints imposed on membrane-spanning helices. Changes in local hydropathicity, average flexibility, protein stability (ΔΔG), hydrogen-bonding patterns, surface hydrophobicity and electrostatic potential distributions were also evaluated. Stability predictions obtained using I-Mutant2.0 and DynaMut2.0 indicated that many substitutions tend to destabilize P-gp, although differences in ΔΔG values were observed between methods due to distinct algorithmic approaches. Structural superposition analyses demonstrated generally minor backbone deviations, yet local alterations in surface hydrophobicity, electrostatic potential, and hydrogen bond networks were evident. These physicochemical perturbations, even when subtle, may influence conformational dynamics and coupling between nucleotide-binding and transmembrane domains. Overall, these findings suggest that single amino acid substitutions may alter the local structural environment of P-gp and could potentially influence its transport function.