Influence of the fluorophore mobility on distance measurements by gas phase FRET.

Abstract

Gas-phase Förster resonance energy transfer (FRET) combines the advantages of mass spectrometry and fluorescence spectroscopy for the conformational analysis of mass-selected biomolecules. While this implementation of FRET in the gas phase promises detailed insights for fundamental and applied studies, the gas-phase environment also poses great challenges. For FRET, fluorophore pairs are typically covalently attached to strategic binding sites in the backbone of a biomolecule, using short linkers. The linker further increases the mobility of the dye, contributing to rotational averaging of the relative orientation of the transition dipole moments of donor and acceptor. However, little is known about the fluorophore’s degrees of freedom in the gas phase and how it may be influenced by intramolecular interactions. In this study, we test the influence of a fluorophore’s linker length on the measured FRET efficiencies in the gas phase to probe the mobility of the fluorophore. An increased FRET efficiency was observed with increasing linker length, ranging from 5.3 % for a linker consisting of 2 atoms to 27.7 % for a linker length of 13 atoms. To rationalize this trend, we profiled the conformational landscape of each model system with MD simulations. Employing state-of-the-art enhanced sampling techniques, we captured intramolecular interactions that promote a population shift towards smaller donor-acceptor separation for longer linker lengths and induce a significant increase in their acceptor dipole. The presented methodology is a first step towards the explicit consideration of a fluoruophore’s range of motion in the interpretation of gas-phase FRET experiments.

Probing the Stability of a β-Hairpin Scaffold after Desolvation

“…Notably also the theoretical R 0 , a fitting parameter, was kept the same. 48 Hence, applying this procedure did not involve any fine-tuning of parameters and is an unbiased way of estimating the FRET efficiencies. As shown in Figure 2a, the predicted FRET efficiencies match the experimental values.…”

“…In their recent work, Metternich et al developed a novel simulation and analysis pipeline for the prediction of FRET efficiencies in the gas phase. 48 Because the system studied here is similar, we were able to use the optimized procedure found by Metternich et al to further investigate GB1p in silico. Specifically, Coulombic replica exchange MD (C-REMD) simulations of the three Cu-bound species were carried out (SI 7).…”