title: Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies.
abstract: Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.
authors.affiliation: Royal Institute of Technology (KTH), Dept Applied Physics, Experimental Biomolecular Physics, Albanova Univ Center, 106 91 Stockholm, Sweden.
authors.forename: Heike
authors.initials: H
authors.lastname: Hevekerl
authors.affiliation: Royal Institute of Technology (KTH), Dept Applied Physics, Experimental Biomolecular Physics, Albanova Univ Center, 106 91 Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Tornmalm
authors.affiliation: Royal Institute of Technology (KTH), Dept Applied Physics, Experimental Biomolecular Physics, Albanova Univ Center, 106 91 Stockholm, Sweden.
authors.forename: Jerker
authors.initials: J
authors.lastname: Widengren
mesh_terms: D005453:Fluorescence; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D014364:Tryptophan