Abstract
Time dependent fluorescence Stokes (emission wavelength) shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluorescence probes, both the efficiency and the wavelength of Trp fluorescence in proteins are highly sensitive to microenvironment, and Stokes shifts can be dominated by the well-known heterogeneous nature of protein structure, leading to what we call pseudo-TDFSS: shifts that arise from differential decay rates of subpopulations. Here we emphasize a novel, general method that obviates pseudo-TDFSS by replacing Trp by 5-fluorotryptophan (5Ftrp), a fluorescent analogue with higher ionization potential and greatly suppressed electron-transfer quenching. 5FTrp slows and suppresses pseudo-TDFSS, thereby providing a clearer view of genuine relaxation caused by solvent and protein response. This procedure is applied to the sweet-tasting protein monellin which has uniquely been the subject of ultrafast studies in two different laboratories (Peon, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10964; Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214) that led to disparate interpretations of a 20 ps transient. They differed because of the pseudo-TDFSS present. The current study exploiting special properties of 5FTrp strongly supports the conclusion that both lifetime heterogeneity-based TDFSS and environment relaxation-based TDFSS are present in monellin and 5FTrp-monellin. The original experiments on monellin were most likely dominated by pseudo-TDFSS, whereas, in the present investigation of 5FTrp-monellin, the TDFSS is dominated by relaxation and any residual pseudo-TDFSS is overwhelmed and/or slowed to irrelevance.