Photo Switching of Protein Dynamical Collectivity

TL;DR

This study reveals that photobleaching of red fluorescent proteins increases the temperature at which their picosecond structural motions become collective, indicating a switch in protein dynamical behavior linked to internal water channels.

Contribution

It demonstrates for the first time that protein dynamical transition temperatures can be switched by photobleaching, highlighting the role of internal water channels in protein motion collectivity.

Findings

Photobleaching raises the protein dynamical turn-on temperature.

Photobleached proteins show increased internal water channels.

Protein motions become more collective after photobleaching.

Abstract

We examine changes in the picosecond structural dynamics with irreversible photobleaching of red fluorescent proteins mCherry, mOrange2 and TagRFP-T. Measurements of the protein dynamical transition using terahertz time-domain spectroscopy show in all cases an increase in the turn-on temperature in the bleached state. The result is surprising given that there is little change in the protein surface, and thus the solvent dynamics held responsible for the transition should not change. A spectral analysis of the measurements guided by quasiharmonic calculations of the protein absorbance reveals that indeed the solvent dynamical turn-on temperature is independent of the thermal stability and photostate however the protein dynamical turn-on temperature shifts to higher temperatures. This is the first demonstration of switching the protein dynamical turn-on temperature with protein functional state. The observed shift in protein dynamical turn-on temperature relative to the solvent indicates an increase in the required mobile waters necessary for the protein picosecond motions: that is these motions are more collective. Melting-point measurements reveal that the photobleached state is more thermally stable and structural analysis of related RFPs shows that there is an increase in internal water channels as well as a more uniform atomic root mean squared displacement. These observations are consistent with previous suggestions that water channels form with extended light excitation providing O2 access to the chromophore and subsequent fluorescence loss. We report that these same channels increase internal coupling enhancing thermal stability and collectivity of the picosecond protein motions. The terahertz spectroscopic characterization of the protein and solvent dynamical onsets can be applied generally to measure changes in collectivity of protein motions.