Effect of Trifluoroethanol on the Tryptophan Side Chain Orientation in the Hydrophobic Core of Troponin C Studied by NMR and Time Resolved Fluorescence

Abstract

Several NMR studies have shown that co-solvent trifluoroethanol (TFE) does not perturb the overall three-dimensional structure of proteins. We have inserted a single tryptophan (F77W) in the hydrophobic core of the N-domain of cardiac troponin C (cNTnC), and determined the structure of the mutant with and without TFE (Julien et al., Protein Sci. 2009 18:1165-74). Interestingly, the position of the tryptophan side chain orientation was shown to be in opposite directions. We have monitored the effect of TFE on the tryptophan rotamer population using 13C-HSQC spectra and used a full lineshape analysis to quantify the rate of the conformational exchange.To further characterize this phenomenon, we have used Time Correlated Single Photon Counting experiments as a function of the TFE concentration. The time dependence could be fitted very well with three lifetimes in the wavelength region from 320 to 380 nm, and global analysis was further used. Addition of TFE (up to 19%) causes a gradual decrease of the lifetimes, due to dynamic quenching with very low quenching constants between kq = 0.1 to 0.01 M-1 ns-1. The amplitude fractions of the lifetimes change upon addition of TFE. At 340 nm, the amplitude fraction of the long lifetime (5.9 ns) increased from 13 to 29%, while that of the middle lifetime (3.3 ns) decreased from 63 to 50%. The short lifetime changed only to a limited extend. These data indicate that the change in the tryptophan indole position (different rotameric state) upon addition of TFE, as observed in NMR, is reflected mainly in the amplitude fractions of the different lifetimes. This is consistent, in this case, with the interpretation of lifetimes linked to rotameric states of tryptophan.