A Structure-Based Simulation Approach for Electron Paramagnetic Resonance Spectra Using Molecular and Stochastic Dynamics Simulations

Abstract

Electron paramagnetic resonance (EPR) spectroscopy using site-directed spin-labeling is an appropriate technique to analyze the structure and dynamics of flexible protein regions as well as protein-protein interactions under native conditions. The analysis of a set of protein mutants with consecutive spin-label positions leads to the identification of secondary and tertiary structure elements. In the first place, continuous-wave EPR spectra reflect the motional freedom of the spin-label specifically linked to a desired site within the protein. EPR spectra calculations based on molecular dynamics (MD) and stochastic dynamics simulations facilitate verification or refinement of predicted computer-aided models of local protein conformations. The presented spectra simulation algorithm implies a specialized in vacuo MD simulation at 600 K with additional restrictions to sample the entire accessible space of the bound spin-label without large temporal effort. It is shown that the distribution of spin-label orientations obtained from such MD simulations at 600 K agrees well with the extrapolated motion behavior during a long timescale MD at 300 K with explicit water. The following potential-dependent stochastic dynamics simulation combines the MD data about the site-specific orientation probabilities of the spin-label with a realistic rotational diffusion coefficient yielding a set of trajectories, each more than 700 ns long, essential to calculate the EPR spectrum. Analyses of a structural model of the loop between helices E and F of bacteriorhodopsin are illustrated to demonstrate the applicability and potentials of the reported simulation approach. Furthermore, effects on the motional freedom of bound spin-labels induced by solubilization of bacteriorhodopsin with Triton X-100 are examined.

Conformational dynamics and distribution of nitroxide spin labels

“…Combinations of these values lead to 108 canonical rotamers. Distributions of dihedral angles observed in MD computations for MTSL attached to the central cysteine residue of a pentadecaglycine helix in explicit water indeed roughly agree with the expected minima, with small deviations of the optimum dihedral angles resulting from steric hindrance by atoms that are remote from the considered dihedral angle [47]. Corresponding plots for MTSL attached to a single cysteine residue, based on data from [41], are shown in Fig.…”

“…However, no such interactions were apparent in standard ab initio quantum chemical parametrization of CHARMM force field parameters for MTSL [52]. As remarked upon earlier [47], sulfur atoms are highly polarizable, so that spherically symmetric Lennard-Jones potentials in standard force fields may not be sufficient to model van-der-Waals interactions, a point that has been reiterated recently [60].…”

“…First, transitions between the p and m states of 3 are too infrequent in MD simulations at ambient temperature to obtain good statistics [49,52]. Frequent transitions are observed at a simulation temperature of 600 K [47];…”

“…Transitions between the p and m states of 3 are particularly slow and are observed only rarely in MD trajectories up to 100 ns at ambient temperature [47][48][49]. Sampling problems were also encountered in an MD study where distance distributions between spin labels were directly taken from MD trajectories and compared to experimental distributions [50].…”

Nanometer‐Scale Water‐ and Proton‐Diffusion Heterogeneities across Water Channels in Polymer Electrolyte Membranes

“…As Nafion is gradually dehydrated, empirically achieved with a set amount of dehydration time at 60°C (see the legend of Figure 1b) for dehydration duration), a new spectral signature appears, indicating less-mobilized 4-hydroxy-TEMPO whose amplitude increases with longer dehydration duration, as marked with an arrow in Figure 1b) at the lower magnetic field region. [18][19][20][21] The EPR spectrum of TEMPO imbibed in fully hydrated Nafion in Figure 1c) can be simulated with a longer τ r ~2.2 ns compared to that of 4-hydroxy-TEMPO, with the result shown in the inset. As with 4-hydroxy-TEMPO, the broad spectral component becomes stronger as the sample is dehydrated, as marked with an arrow in Figure 1c).…”