In Silico Functional Annotation and Structural Characterization of Hypothetical Proteins in Bacillus paralicheniformis and Bacillus subtilis Isolated from Honey

Bacillus species are ubiquitous and survive in competitive microbial communities under adverse environmental conditions. Bacillus paralicheniformis and Bacillus subtilis obtained from honey revealed a significant proportion of proteins within their genomes as uncharacterized hypothetical proteins (HPs). A total of 1007 HP sequences were evaluated, resulting in the successful annotation of 56 HPs by assigning specific functions to them. A systematic in silico approach, integrating a range of bioinformatics tools and databases to annotate functions, characterize physicochemical properties, determine subcellular localization, and study protein–protein interactions, was used. Homology and de novo models were generated for the HPs, coupled with iterative remodeling and molecular dynamics (MD) simulations. HPs having significant roles in sporulation, biofilm formation, motility, ion transportation, regulation of metabolic processes, DNA repair, replication, and transcription were identified. Classical MD simulations of globular and transducer membrane proteins, along with postprocessing analyses, refined our structural predictions and provided deeper insights into the stability and functional dynamics of the protein structures under physiological conditions. Moreover, we observed a correlation between the percentage of α helix, β sheet, and coil structures in globular proteins and transducer membrane proteins. The integration of iterative loop modeling, MD simulations, and Dictionary of Secondary Structure in Proteins analysis further validated our predicted models and facilitated the identification of regions critical for protein function, thereby enhancing the overall reliability and robustness of our functional annotations. Furthermore, annotation of these hypothetical proteins aids in identifying novel proteins within bacterial cells, ultimately contributing to a deeper understanding of bacterial cell biology and their use for biotechnological purposes.