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Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements.

Klose, D; Klare, JP; Grohmann, D; Kay, CW; Werner, F; Steinhoff, HJ; (2012) Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements. PLOS One , 7 (6) , Article e39492. 10.1371/journal.pone.0039492. Green open access

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Abstract

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.

Type: Article
Title: Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements.
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0039492
Publisher version: http://dx.doi.org/10.1371/journal.pone.0039492
Language: English
Additional information: © 2012 Klose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Work in the RNAP laboratory was supported by Wellcome Trust (www.wellcome.ac.uk) grant 079351/Z/06/Z and BBSRC (www.bbsrc.ac.uk) grants BB/E008232/1 and BB/H019332/1 to FW. DK received support from grants DFG (www.dfg.de) SFB 431 P18 and DFG SFB 944 P10 to H-JS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology
URI: http://discovery.ucl.ac.uk/id/eprint/1361556
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