TDSS in Trp Fluorescence Reveals Multiple Protein and Solvent Relaxation Modes

Abstract

The Time-Dependent Spectral Shifts (TDSS) in the fluorescence of solvatochromic dyes in polar solvents report solvent relaxation dynamics, which in water occurs on the femtosecond timescale. The TDSS in the emission of tryptophan and other solvatochomic fluorophores in proteins span a range of timescales from femtoseconds to nanoseconds. MD simulations of the GB1 protein in TIP3P water made it possible to separate five relaxation modes and to explain their physical origins. Two of these relaxation modes also contribute to the TDSS of dyes in polar solvents. The ultrafast relaxation mode (τ∼35fs) is due in part to the librational relaxation of water molecules and in part to the small adjustments in the local protein structure. This mode is responsible for about half of the total TDSS amplitude. Two collective rotational relaxation modes of water molecules are known. The longitudinal (τL∼550fs) mode contributes to the TDSS of both dyes in water and tryptophan residues in proteins. The transverse (τD∼8.3ps) relaxation cannot contribute to the TDSS of dyes in water, but it contributes to the TDSS in proteins having internal water channels or pockets. This can be used to study internal water. In MD simulations using TIP3P water both τL and τD are ∼30% shorter than the experimental values. A small (<0.25Å) adjustment of the GB1 tertiary structure occurs in MD simulations on the time scale of 130ps and results in a 140cm−1 contribution to the TDSS. A shift in the sidechain conformation of Glu-42 in close proximity to the fluorophore (Trp-43) is the main contributor to the slow TDSS amplitude (470cm−1). This conformational change takes 2.6ns in MD simulations and only 80ps in the experiment, which reveals that in CHARMM22 the potential barriers separating sidechain conformations are too high.