Protein–Bath Coupling of an Internal Reaction Coordinate at Intermediate Time Scales

Abstract

Thermally activated barrier-crossing processes are central to protein reaction kinetics. A determining factor for such kinetics is the extent to which the protein's motions are coupled to the surrounding bath. It is understood that slow large-scale conformational motions are strongly coupled to the environment, while fast librational motions are uncoupled. However, less is known about protein−bath coupling of reaction coordinates located on the interior of a protein and with dynamics on intermediate time scales. In this work, we use single molecule 2D fluorescence lifetime correlation spectroscopy to study the microsecond chemical reaction occurring in the chromophore pocket of eGFP. The equilibrium reaction involves a dihedral rotation of a glutamic acid residue and a rearrangement of the local hydrogen-bonding network surrounding the endogenous chromophore, with no accompanying large-scale conformational changes. We observe that the internal chemical reaction is coupled to the solvent viscosity, though the scaling deviates from Kramers' behavior. We attribute this deviation to the internal friction of the protein, which weakens the protein−solvent coupling at high viscosity and intermediate time scales.

Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

“…As an alternative to smFRET experiments, we developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) and reported it in 2013. , 2D FLCS utilizes the fluorescence lifetime information to distinguish different fluorescence species in a sample without any a priori knowledge and detects their interconversion dynamics with submicrosecond resolution. , The time resolution of 2D FLCS is two or 3 orders of magnitude higher than smFRET, enabling the detection of rapid conformational changes on the microsecond-millisecond time scale. − In the original 2D FLCS, we used the one-color excitation and one-color fluorescence detection scheme to distinguish different fluorescence species based on the donor fluorescence lifetime (single-color 2D FLCS). − Recently, we extended 2D FLCS by introducing acceptor fluorescence information, i.e., two-color (detection) 2D FLCS, in which the donor is excited and time-resolved fluorescence signals from both the donor and acceptor are recorded. , Two-color 2D FLCS provides significantly better sensitivity for high-FRET species that only exhibit a weak donor fluorescence (but a strong acceptor fluorescence) compared to single-color 2D FLCS that only detects the donor fluorescence. Nevertheless, it remains challenging to distinguish a low- or zero-FRET species from the D-only species that very often coexist in sample solutions.…”

Viscoelasticity of single folded proteins using dynamic atomic force microscopy

“…In all these experiments the friction is measured in an indirect manner and its usefulness is debated in the literature. [34][35][36][37] In contrast to indirect ways of measuring stiffness, Wang and Zocchi measured the viscoelasticity of folded domains of guanylate kinase by applying sinusoidal stress to protein coated Au nanoparticles and measuring the corresponding strain by evanescent wave scattering. 19,38 By modelling folded domains as Maxwell's viscoelastic element -a spring and dash-pot in series -the stiffness and damping coefficient are inferred by fitting data to the observed amplitude-response curve.…”

“…In all these experiments the friction is measured in an indirect manner and its usefulness is debated in the literature. 34–37…”