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ABSTRACT: The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.
PLoS Computational Biology 09/2012; 8(9):e1002683. · 5.22 Impact Factor
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ABSTRACT: Fluorescence resonance energy transfer (FRET) can be utilized to gain low-resolution structural information by reporting on the proximity of molecules or measuring inter- and intramolecular distances. This method exploits the fact that the probability of the energy transfer is related to the separation between the fluorescent molecules. This relationship is well described for a single pair of fluorophores but is complicated in systems containing more than two fluorophores. Here, we present a Monte Carlo calculation scheme that has been implemented through a user-friendly web-based program called ExiFRET that can be used to determine the FRET efficiency in a wide range of fluorophore arrangements. ExiFRET is useful to model FRET for individual fluorophores randomly distributed in two or three dimensions, fluorophores linked in pairs or arranged in regular geometries with or without predefined stoichiometries. ExiFRET can model both uniform distributions and fluorophores that are aggregated in clusters. We demonstrate how this tool can be employed to understand the effect of labeling efficiency on FRET efficiency, estimate relative contributions of inter- and intramolecular FRET, investigate the structure of multimeric proteins, stoichiometries, and oligomers, and to aid experiments studying the aggregation of lipids and proteins in membrane environments. We also present an extension that can be used to study instances in which fluorophores have constrained orientations.
Journal of Biomedical Optics 01/2012; 17(1):011005. · 3.16 Impact Factor
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ABSTRACT: Fluorescence resonance energy transfer (FRET) is commonly used to determine the proximity of fluorophores, but usually many assumptions are required to gain a quantitative relationship between the likelihood of energy transfer and fluorophore separation. Molecular Dynamics (MD) simulations provide one way of checking these assumptions, but before using simulations to study complex systems it is important to make sure that they can correctly model the motions of fluorophores and the likely FRET efficiency in a simple system. Here we simulate a well characterised situation of independent fluorophores in solution so that we can compare the predictions with expected values. Our simulations reproduce the experimental fluorescence anisotropy of Alexafluor488 and predict that of AlexaFluor568. At the ensemble level we are able to reproduce the expected isotropic and dynamic motion of the fluorophores as well as the FRET efficiency of the system. At the level of single donor-acceptor pairs, however, very long simulations are required to adequately sample the translational motion of the fluorophores and more surprisingly also the rotational motion. Our studies demonstrate how MD simulations can be used in more complex systems to check if the dynamic orientation averaging regime applies, if the fluorophores have isotropic orientational motion, to calculate the likely values of the orientation factor κ(2) and to determine the FRET efficiency of the system in both dynamic and static orientational averaging regimes. We also show that it is possible in some situations to create system specific relationships between FRET efficiency and fluorophore separation that can be used to interpret experimental data and find any correlations between κ(2) and separation that may influence distance measurements.
Physical Chemistry Chemical Physics 06/2011; 13(23):11045-54. · 3.57 Impact Factor
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ABSTRACT: We discuss a generalization of the resolution of the identity by considering one-body resolutions of two-body operators, with particular emphasis on the Coulomb operator. We introduce a set of functions that are orthonormal with respect to 1r(12) and propose that the resulting "resolution of the Coulomb operator," r(12) (-1)=mid R:phi(i)phi(i)mid R:, may be useful for the treatment of large systems due to the separation of two-body interactions. We validate our approach by using it to compute the Coulomb energy of large systems of point charges.
The Journal of chemical physics 06/2008; 128(20):201104. · 3.09 Impact Factor
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