title: Utility of 5-Cyanotryptophan Fluorescence as a Sensitive Probe of Protein Hydration.
abstract: Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics, and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein's hydration status and dynamics. Herein, we show that a tryptophan analogue, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN-containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.
authors.affiliation: Department of Chemistry and ‡The Ultrafast Optical Processes Laboratory, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Beatrice N
authors.initials: BN
authors.lastname: Markiewicz
authors.affiliation: Department of Chemistry and ‡The Ultrafast Optical Processes Laboratory, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Debopreeti
authors.initials: D
authors.lastname: Mukherjee
authors.affiliation: Department of Chemistry and ‡The Ultrafast Optical Processes Laboratory, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Troxler
authors.affiliation: Department of Chemistry and ‡The Ultrafast Optical Processes Laboratory, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Gai
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014867:Water / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D014867:Water; D014364:Tryptophan