title: Tryptophan side chain conformers monitored by NMR and time-resolved fluorescence spectroscopies.
abstract: We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Julien
authors.forename: Guoli
authors.initials: G
authors.lastname: Wang
authors.forename: Abel
authors.initials: A
authors.lastname: Jonckheer
authors.forename: Yves
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: Brian D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000737:chemistry; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D012997:Solvents; D019209:Troponin C; D014270:Trifluoroethanol; D014364:Tryptophan