Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

Abstract

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET) provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for novel Fe2+- and Ru2+-based tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET

“…To investigate the utility of time-resolved FRET to measure distance distributions, we incorporated the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) into MBP using amber codon suppression in bacteria as previously described (12,28,29). MBP is a clamshell-shaped protein that undergoes a significant closure of the clamshell upon binding maltose.…”

“…This paper applies time-resolved tmFRET to study protein allostery and conformational dynamics. tmFRET utilizes a fluorescent noncanonical amino acid as the donor and our new metal-bipyridyl derivatives as the acceptor to overcome limitations of traditional FRET methods (11). We applied this method to MBP and demonstrated it can accurately determine distances, conformational heterogeneity, and energetics.…”

“…τ D1 , τ D2 , and R 0 were constrained to previously determined values, f D , r̅ 1 , σ 1 , r̅ 2 , σ 2 , f B were varied but constrained to be the same across all maltose concentrations, and A 2 and t 0 were varied separately for each maltose concentration. Occasionally, if f D ,was inconsistent between individual fits of the same experiment, it was held at 0.1 as previously determined (11). Under these conditions, the values of the parameters were well determined (Figure S2) and independent of the initial guesses (Figure S3).…”

“…For tmFRET experiments, the expression and purification of MBP was done as described previously (11). Briefly, MBP TAG constructs with a C-terminal twin-strep tag were cotransfected with a plasmid containing the AcdA9 aminoacyl tRNA synthetase and its cognate tRNA (12) in BL-21(DE3) cells.…”

“…We recently developed a novel system for time-resolved FRET that overcomes these limitations by combining a noncanonical amino acid fluorophore donor and a transition metal ion acceptor (10). Here we employ this novel system with metal-bipyridyl acceptors, developed in a companion paper in this issue (11), to measure longer distance distributions in a model protein, maltose binding protein (MBP). We show that time-resolved FRET can quantify both the heterogeneity of a given conformational state and the energetics that govern the distribution of a protein among conformational states, collectively referred to as protein dynamics.…”