An integrative multitiered computational analysis for better understanding the structure and function of 85 miniproteins

Miniproteins, defined as polypeptides containing fewer than 50 amino acids, have recently elicited significant interest due to an emerging understanding of their diverse roles in fundamental biological processes. In addition, miniprotein dysregulation underlies human diseases and is a considerable focus for biotechnology and drug development. The human genome project revealed many miniproteins, most of which remain uncharacterized. This study reports an approach for analyzing and scoring previously uncharacterized miniproteins by integrating knowledge from classic sequence-based bioinformatics, computational biophysics, and system biology annotations. We identified 85 human miniproteins using this simple multi-tier approach. Then, we predicted miniprotein three-dimensional structures using AI-based methods and peptide modeling to determine their relative yields for these understudied polymers. We identify that structural propensity is not strictly dependent on polymer length, and peptide-based algorithms may have advantages over AI-based algorithms for certain groups of miniproteins. Subsequently, we used several computational biophysics methods and structure-based calculations to annotate and evaluate results from both algorithms. We propose novel structure-function relationships for miniproteins, which expands our understanding of their potential roles in cellular processes. Finally, we practically identify which sequence- and structure-based tools provide the most information, aiding future studies of miniproteins, with emphasis on their biomedical relevance.