Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

Abstract

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision

“…Along these lines, we would like to point out that the LD555p and LD655 fluorophores have long (13 atoms) linkers to minimize any possible restrictions on fluorophore tumbling and a number of polar groups to minimize any possible hydrophobic interactions with the protein. Explicit solvent, full potential MD simulations with these fluorophores attached to a protein of similar size to barr1 showed minimal protein-fluorophore interactions and estimated k 2 to be near to the ideal value (Girodat et al, 2020).…”

“…Finally, traces were corrected for donor to acceptor channel crosstalk, unequal apparent brightness of donor and acceptor fluorophores, and acceptor direct excitation (Hellenkamp et al, 2018). Corrected FRET values (E) were then used for estimating inter-dye distances (R) using the following equation and an R 0 of 62 A ˚ (Girodat et al, 2020).…”

“…The resulting ensemble of fluorophore positions serves as an approximation to the experimental ensemble that gives rise to the observed FRET efficiencies. While these simplified, structure-based potential simulations do not capture all contributors to fluorophore positions and dynamics, they have been shown to provide good correspondence to smFRET experiments with a protein of similar size to barr1 (Girodat et al, 2020).…”

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation

“…From these two measurements, the orientation of the absorption transition dipole relative to the origami structure is obtained as (further details in Supplementary Note 1). Taking into account the dipolar symmetry and that the lowest electronic transition in carbocyanine dyes is polarized along the principal axis of the molecule, − ρ reports the in-plane projection of the molecular orientation as depicted in Figure a. Due to the symmetry of the DNA PAINT sites, we constrain our results in the range ρ ∈ [0, 90°], which is the relevant angle to characterize most orientation-dependent molecular interactions.…”

Out-of-Equilibrium Biophysical Chemistry: The Case for Multidimensional, Integrated Single-Molecule Approaches

“…smFRET used in combination with MD simulations bridged multiple length and time scale limitations associated with each technique independently. The combination has been used to validate the leucine–isoleucine–valine binding protein (LIV-BP) as a biosensor, investigate rapid dynamics along the reaction coordinate, capture dynamic binding and allosteric processes, quantify supertertiary and transient conformations, and probe the equilibrium dynamics for biomolecular states. ,, Additionally, optical tweezers used in combination with multiscale molecular dynamics simulations have characterized the effects of octanoyl-CoA on the folding stability of acyl-CoA binding protein and revealed how disease-causing mutations in kinesin-3 motors affect force generation in one dimension through allosteric effects on the ATP hydrolysis site in another dimension of its multidimensional energy landscape . Single-molecule fluorescence spectroscopy combined with force spectroscopy approaches provided mechanistic details for force-induced changes and local conformational dynamics in DNA nanostructures and out-of-equilibrium conformational dynamics in the protein–DNA interactions of the E. coli DNA repair helicase (UvrD) system .…”