Pro-SMP finder-A systematic approach for discovering small membrane proteins in prokaryotes

Prokaryotic chromosomes contain numerous small open reading frames (ORFs) of less than 200 bases. Since high-throughput proteomics methods often miss proteins containing fewer than 60 amino acids, it is difficult to decern if they encode proteins. Recent studies have revealed that many small proteins are membrane proteins with a single membrane-anchoring α-helix. As membrane anchoring or transmembrane motifs are accurately identifiable with high confidence using computational algorithms like Phobius and TMHMM, small membrane proteins (SMPS) can be predicted with high accuracy. This study employed a systematic approach, utilizing well-verified algorithms such as Orfipy, Phobius, and Blast to identify SMPs in prokaryotic organisms. Our main search parameters targeted candidate SMPs with an open reading frame between 60-180 nucleotides, a membrane-anchoring or transmembrane region 15 and 30 amino acids long, and sequence conservation among other microorganisms. Our findings indicate that each prokaryote possesses many SMPs, with some identified in the intergenic regions of currently annotated chromosomes. More extensively studied microorganisms, such as Escherichia coli and Bacillus subtilis, have more SMPs identified in their genomes compared to less studied microorganisms, suggesting the possibility of undiscovered SMPs in less studied microorganisms. In this study, we describe the common SMPs identified across various microorganisms and explore their biological roles. We have also developed a software pipeline and an accompanying online interface for discovering SMPs (http://cs.indstate.edu/pro-smp-finder). This resource aims to assist researchers in identifying new SMPs encoded in microbial genomes of interest.