2.3 Evolutionary conservation, surface accessibility, and post-translational modification (PTM) analysis

The degree to which an amino acid position is evolutionarily conserved, reflects its structural and functional importance. Therefore, the conservancy of each position in the amino acid sequence of the mTOR protein was analyzed by the ConSurf server (https://consurf.tau.ac.il/) [37]. This tool estimates the evolutionary rate of each amino acid position based on the phylogenetic relations between homologous sequences. The software can correctly distinguish genuine sequence conservation from other conservation that can result from a short evolutionary time. In this study, the mTOR protein sequence was analyzed against the UNIREF-90 protein database using the HMMER homolog search algorithm with a 0.0001 E-value cut-off and 3 HMMER iterations.

To determine the surface accessibility of each amino acid residue of mTOR protein, NetSurfP-2.0 (https://services.healthtech.dtu.dk/service.php?NetSurfP-2.0) was used [38]. This web tool is sequence-based and uses neural networks to predict several local structural features including solvent accessibility, secondary structure, and structural disorder for each residue of the query sequences.

Post-translational modifications within a protein molecule are also crucial. They introduce new functionalities and exert control over protein structure and function by modulating intra- and intermolecular interactions. In this study, the web-based tool MusiteDeep (https://www.musite.net/) was applied to predict potential PTM sites and corresponding probable modifications in the mTOR protein sequence [39]. Rather than predicting only a single type of PTM, this web server provides a deep-learning framework for general protein PTM site prediction and visualization using raw protein sequences as input.