EFFECTS OF MUTATIONS AT POSITION 161 ON FLUORESCENCE LOSS IN mCHERRY

DOI: 10.18173/2354-1059.2025-0025

Abstract

mCherry is among the most widely used red fluorescent proteins in advanced imaging techniques. However, recent studies have revealed that mCherry and its variants are susceptible to spontaneous degradation, leading to a gradual loss of fluorescence over time. In this study, six mCherry variants were engineered, each featuring a single amino acid mutation at position 161, to investigate the role of this residue in mCherry fluorescence stability. Spectroscopic analysis demonstrated that these substitutions had variable effects on fluorescence retention. Notably, the substitution of Ile161 with Ser, Thr, and Gly resulted in a pronounced loss of fluorescence, whereas replacement with Ala, Val, and Cys produced milder effects. These findings underscore the critical role of residue 161 in chromophore stability in red fluorescent proteins, offering valuable suggestions for designing more stable mCherry variants. Furthermore, preliminary crystallization trials identified promising conditions for future optimization and structural studies, which may elucidate the molecular basis of substitution influence at position 161 on fluorescence stability in mCherry variants.