Trp Fluorescence in GB1: Nanosecond Dynamics Strongly Depend on pH While 30Ps Relaxation is Constant

Abstract

The fluorescence dynamic Stokes shift(FDSS)of W43 in the model protein GB1, was measured in both picosecond and nanosecond time domains by combining Fluorescence Upconversion with Time Correlated Single Photon Counting (TCSPC). We examined a wide range of pH values (from 8 to 3) where GB1 is known from NMR to be stable. We observe large changes in the nanosecond lifetimes and DAS; Trp lifetime declines with reduced pH, likely due to differences in the Trp charge environment. Stern-Volmer plots revealed changes in Trp exposure. In the picosecond domain, however, a characteristic risetime of 30 ps was seen for GB1 at 375nm. This negative amplitude remains constant throughout the titration, independent of the nanosecond fluorescence changes of W43. This leads us to believe that the W43 rise is not reporting simply the dynamics of the first hydration layer, but instead upon longer range solvation.To rationalize these observations, three sets of 300 2-ns MD+QM simulations were carried out for different levels of protonation of glutamate and aspartate residues. During simulations Trp is switched from ground charges to 1La charges, and the average fluorescence wavelength was computed vs. time. Early results are consistent with the observation of a ∼30 ps FDSS component, independent of pH. The protonated carboxyl group of Glu27 is solvated and intermittently comes in close contact with the Trp ring, strongly suggesting that electron transfer to this group causes ns lifetime shortening at lower pH. The trajectories also show that Lys31 and Glu27 lie salt-bridged above the Trp pyrrole ring at high pH. The blue shifting of the emission at lower pH is consistent with the Lys31 positive charge over the pyrrole ring at low pH.