title: Reduced fluorescence lifetime heterogeneity of 5-fluorotryptophan in comparison to tryptophan in proteins: implication for resonance energy transfer experiments.
abstract: Tryptophan (Trp), an intrinsically fluorescent residue of proteins, has been used widely as an energy donor in fluorescence resonance energy transfer (FRET) experiments aimed at measuring intramolecular distances and distance distributions in protein folding-unfolding reactions. However, the high level of heterogeneity associated with the fluorescence lifetime of tryptophan, even in single-tryptophan proteins, imposes restrictions on its use as the energy donor. A search for a tryptophan analogue having reduced lifetime heterogeneity when compared to tryptophan led us to 5-fluorotryptophan (5F-Trp). A single tryptophan-containing mutant form of barstar, a small 89-residue bacterial protein, has multiple lifetime components in its various structural forms including the unfolded state, similar to observations made with several other proteins. Biosynthetic incorporation of 5F-Trp in place of Trp in the mutant barstar resulted in a significant decrease in the level of heterogeneity of fluorescence decay when compared to Trp-barstar, in the native state as well as in the denatured state. Importantly, observation of a major decay component of more than 80% in both the states makes 5F-Trp a significantly better candidate for being the energy donor in FRET experiments, as compared to Trp. This is expected to enable an unambiguous estimation of intramolecular distance distributions during protein folding and unfolding. The sequence insensitivity of the fluorescence decay kinetics of 5F-Trp in proteins was demonstrated by observing the decay kinetics of 5F-Trp incorporated in several synthetic peptides.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Saswata Sankar
authors.initials: SS
authors.lastname: Sarkar
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
authors.forename: G
authors.initials: G
authors.lastname: Krishnamoorthy
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D019277:Entropy; D031541:Fluorescence Resonance Energy Transfer; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C026927:5-fluorotryptophan; C057438:barstar protein, Bacillus amyloliquefaciens; D014364:Tryptophan