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Multifrequency electron spin resonance spectra of a spin-labeled protein calculated from molecular dynamics simulations.

ABSTRACT: Multifrequency electron spin resonance (ESR) spectra provide a wealth of structural and dynamic information about the local environment of the spin label and, indirectly, about the spin-labeled protein. Relating the features of the observed spectra to the underlying molecular motions and interactions is, however, challenging. To make progress toward a rigorous interpretation of ESR spectra, we perform extensive molecular dynamics (MD) simulations of fully solvated T4 Lysozyme, labeled with the spin label MTSSL at positions 72 and 131. These two sites have been the object of numerous experimental studies and are generally considered as prototypical solvent-exposed sites on the surfaces of alpha-helices. To extend the time window afforded by the MD simulations, stochastic Markov models reflecting the dynamics of the spin label side chains in terms of their rotameric states are constructed from the trajectories. The calculated multifrequency ESR spectra are in very good agreement with experiment for three different magnetic field strengths without adjusting any parameters. During the trajectories, the spin labels interconvert among a fairly large number of conformations and display a propensity to form interactions with protein residues other than their nearest neighbors along the helix. The detailed picture of the spin label emerging from the MD simulations provides useful insight into the molecular origins of the available spectroscopic and crystallographic data.

SUBMITTER: Sezer D

PROVIDER: S-EPMC2763593 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Multifrequency electron spin resonance spectra of a spin-labeled protein calculated from molecular dynamics simulations.

Sezer Deniz D Freed Jack H JH Roux Benoît B

Journal of the American Chemical Society 20090201 7

Multifrequency electron spin resonance (ESR) spectra provide a wealth of structural and dynamic information about the local environment of the spin label and, indirectly, about the spin-labeled protein. Relating the features of the observed spectra to the underlying molecular motions and interactions is, however, challenging. To make progress toward a rigorous interpretation of ESR spectra, we perform extensive molecular dynamics (MD) simulations of fully solvated T4 Lysozyme, labeled with the s ...[more]

PMID: 19191603

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Project description:An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

Project description:Spin-labeled lipids are commonly used as fluorescence quenchers in studies of membrane penetration of dye-labeled proteins and peptides using depth-dependent quenching. Accurate calculations of depth of the fluorophore rely on the use of several spin labels placed in the membrane at various positions. The depth of the quenchers (spin probes) has to be determined independently; however, experimental determination of transverse distributions of spin probe depths is difficult. In this Article, we use molecular dynamics (MD) simulations to study the membrane behavior and depth distributions of spin-labeled phospholipids in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. To probe different depths within the bilayer, a series containing five Doxyl-labeled lipids (n-Doxyl PC) has been studied, in which a spin moiety was covalently attached to nth carbon atoms (where n = 5, 7, 10, 12, and 14) of the sn-2 stearoyl chain of the host phospholipid. Our results demonstrate that the chain-attached spin labels are broadly distributed across the model membrane and their environment is characterized by a high degree of mobility and structural heterogeneity. Despite the high thermal disorder, the depth distributions of the Doxyl labels were found to correlate well with their attachment positions, indicating that the distribution of the spin label within the model membrane is dictated by the depth of the nth lipid carbon atom and not by intrinsic properties of the label. In contrast, a much broader and heterogeneous distribution was observed for a headgroup-attached Tempo spin label of Tempo-PC lipids. MD simulations reveal that, due to the hydrophobic nature, a Tempo moiety favors partitioning from the headgroup region deeper into the membrane. Depending on the concentration of Tempo-PC lipids, the probable depth of the Tempo moiety could span a range from 14.4 to 18.2 Å from the membrane center. Comparison of the MD-estimated immersion depths of Tempo and n-Doxyl labels with their suggested experimental depth positions allows us to review critically the possible sources of error in depth-dependent fluorescence quenching studies.

Project description:The molecular dynamics of spin-labeled compounds included into the solid phase of cyclodextrins (CDs) has been studied using conventional (X-band) ESR at 9 GHz and high-field high-frequency (HFHF) ESR at 240 and 170 GHz. The patterns of axial rotation at these higher frequencies are clear just by inspection of the spectrum, unlike the case for 9 GHz spectra. That is HFHF ESR is sensitive to molecular motion about the diffusion axis collinear with the X, Y or Z-direction of the magnetic g- and A-tensors of the nitroxide moiety (referred to, respectively, as X, Y or Z-rotation). For doxyl stearic acids (Z-rotation) and TEMPOyl caprylate (X-rotation) included in beta- and gamma-CDs we were able to determine the rate of molecular motion and the corresponding potential barriers. We emphasize that determining the rate of Z-rotation by ESR is feasible only using HFHF ESR. For the X-rotation case we suggest that the motion of the nitroxide moiety consists of fast small-angle librations about the magnetic X-axis superimposed by rotational diffusion about the same axis. The potential barrier of 1.7 Kcal mol(-1) for this rotational diffusion is unusually low. A fascinating feature of TEMPO derivatives included in beta-CD is the detectable molecular motion at temperatures below 77 K. For the other CD-spin probe systems, we used multifrequency analysis to assign the conformations of spin-labeled molecules. A dramatic spectral change for 16-sasl in beta- and gamma-CDs at approximately 260 K corresponds to a tilting of the position of the nitroxide moiety on the rotating molecule relative to the long diffusion axis, while for TEMPO derivatives in gamma-cyclodextrin below 200 K, we observe a rapid transition from fast to very slow rotational motion. More complex features are best studied by means of multifrequency ESR experiments. The visual clarity and the simplicity of analysis of the ESR spectra shown in this work should provide a benchmark for future studies of molecular motion by HFHF ESR.

Dataset Information

Multifrequency electron spin resonance spectra of a spin-labeled protein calculated from molecular dynamics simulations.

Publications

Multifrequency electron spin resonance spectra of a spin-labeled protein calculated from molecular dynamics simulations.

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