Structural highlights
Publication Abstract from PubMedStructural dynamics play a crucial role in protein function, and tuning these dynamics through mutagenesis has emerged as a promising strategy for enhancing activity. However, identifying dynamics hotspots for protein engineering remains a labor-intensive challenge. Here, we demonstrate that NMR peak intensity analysis-a rapid, qualitative method with residue-level resolution-can identify functionally relevant dynamic regions with high precision. Using a family of red fluorescent proteins (RFPs) as a case study, we reveal that flexibility in specific regions of their structures correlates with function. Specifically, as quantum yield increases, the side of the beta-barrel closest to the chromophore phenolate moiety becomes more rigid, while the opposite side, closest to the acylimine group, gains flexibility. Notably, the phenolate face corresponds to a mutational hotspot frequently targeted in directed evolution campaigns aimed at enhancing brightness, underscoring its functional significance. B-factor analysis of non-cryogenic X-ray crystal structures further supports our findings. Our results establish NMR peak intensity analysis as a promising tool for mapping functional dynamics hotspots to guide protein engineering campaigns. Mapping functional dynamics hotspots for protein engineering with NMR peak intensity analysis.,Damry AM, Hunt SE, Legault S, Thompson MC, Goto NK, Chica RA Protein Eng Des Sel. 2026 Jan 9;39:gzag014. doi: 10.1093/protein/gzag014. PMID:42402021[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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