Novel hybrid protein scaffold gap filling using weighted machine learning ensemble, beam search, and mass-constrained reranking

Abstract

Protein scaffold gap filling is an important computational task in protein sequence reconstruction, where missing amino acid regions must be inferred from incomplete scaffold information. This study proposes a hybrid machine learning and mass constrained reranking framework for protein scaffold gap filling under known-gap-size and known-gapmass settings. Homologous protein sequences from MabCampath, P5A proteoform, and carbonic anhydrase 2 were used to generate masked 11-mer residue-level samples and fullgap evaluation cases. The residue prediction task was formulated as a 20-class amino acid classification problem using first-, middle-, and last-position masking. Multiple classical machine learning models were trained using raw encoded, row-average, and SVD-reduced features, and the strongest models were combined through a validation-accuracy-weighted ensemble. For known-size gap reconstruction, beam search was used to generate complete missing peptide sequences from residue-level probability estimates. For known-mass reconstruction, mass-constrained homologous candidate retrieval was combined with hybrid reranking based on mass validity, homologous frequency, context support, ensemble likelihood, mass error, and length penalty. The proposed framework achieved 95.41% residue-level validation accuracy, 87.50% known-size exact-match accuracy, and 100% top-5 recovery on seven CAH2 known-mass benchmark cases. These results indicate that the proposed framework can effectively reconstruct missing protein regions by integrating local sequence learning, homologous evidence, peptide mass constraints, and biochemical validation.