Integrative Structural Bioinformatics and Molecular Dynamics Analyses of Synaptic Vesicle Proteins and Their Water-Soluble QTY-Variants Reveal Membrane Constraints and Evolutionary Coupling of T ⇔ V.

Synaptic vesicle proteins, including the synaptophysin, synaptogyrins, and synaptic vesicle glycoprotein 2 family are fundamental for neurotransmitter release and synaptic function, influencing numerous physiological processes. Although these proteins hold promise as therapeutic targets, their study has remained complicated due to their location within the cell membrane. To tackle this, we performed comparative analyses on these proteins and their water-soluble variants, which were designed using the QTY code. This approach involves systematically replacing hydrophobic amino acids L (leucine), V/I (valine/isoleucine), and F (phenylalanine) with hydrophilic amino acids Q (glutamine), T (threonine), and Y (tyrosine). The water-soluble QTY variants generated in our study, despite having significant differences in their transmembrane sequences up to 55%, maintained structural similarity, with RMSD values below 1.9 Å. We performed 100 ns molecular dynamics simulations to evaluate the structural stability and conformational dynamics of native and QTY variants. The analysis revealed that QTY substitutions preserved overall fold integrity while inducing localized flexibility, with lipid interactions contributing nonlinear effects that modulate residue-specific dynamics and evolutionary constraints. Our study further identified 155 single nucleotide variants (SNVs) in human genomic databases, and we examined their phenotypic and topological properties. By integrating evolutionary statistics, we provided insights into the substitutional dynamics of hydrophilic and hydrophobic alpha-helices. Notably, we found a strong evolutionary relationship between threonine and valine frequencies in homologous sequences. This coupling persists despite the high mutational barrier requiring a double-nucleotide change, suggesting a functional evolutionary necessity. Our data suggest that QTY variants of synaptic vesicle proteins could be valuable for research in structural biology, evolutionary studies, and medicine, potentially leading to innovative therapeutic strategies for a range of conditions.