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dc.contributor.authorAckermann, Katrin
dc.contributor.authorPliotas, Christos
dc.contributor.authorValera, Silvia
dc.contributor.authorNaismith, Jim
dc.contributor.authorBode, Bela Ernest
dc.date.accessioned2017-11-09T15:30:09Z
dc.date.available2017-11-09T15:30:09Z
dc.date.issued2017-11-07
dc.identifier.citationAckermann , K , Pliotas , C , Valera , S , Naismith , J & Bode , B E 2017 , ' Sparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channels ' , Biophysical Journal , vol. 113 , no. 9 , pp. 1968–1978 . https://doi.org/10.1016/j.bpj.2017.09.005en
dc.identifier.issn0006-3495
dc.identifier.otherPURE: 251009499
dc.identifier.otherPURE UUID: 1495c42d-c837-4619-9003-41e9b6c4731f
dc.identifier.otherScopus: 85032971463
dc.identifier.otherORCID: /0000-0002-4309-4858/work/38548531
dc.identifier.otherORCID: /0000-0002-3384-271X/work/38548532
dc.identifier.otherWOS: 000416212300010
dc.identifier.urihttp://hdl.handle.net/10023/12038
dc.descriptionBEB is grateful for funding from the European Union (Marie Curie Actions REA 334496). This work was supported by the EPSRC (EP/M024660/1) and the Wellcome Trust (099149/Z/12/Z). CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government.en
dc.description.abstractPulse EPR is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g. homo-multimers) due to distorting multi-spin effects. Without suppression of these effects much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multi-spin problem.Here, two different appro aches for suppressing multi-spin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both, the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of E. coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multi-spin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analogue.
dc.language.isoeng
dc.relation.ispartofBiophysical Journalen
dc.rightsCopyright 2017 Biophysical Society. This is an open access article under the CC BY attribution license (http://creativecommons.org/licenses/by/4.0/).en
dc.subjectQD Chemistryen
dc.subjectQH301 Biologyen
dc.subjectDASen
dc.subjectBDCen
dc.subject.lccQDen
dc.subject.lccQH301en
dc.titleSparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channelsen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews.School of Chemistryen
dc.contributor.institutionUniversity of St Andrews.School of Biologyen
dc.contributor.institutionUniversity of St Andrews.EaSTCHEMen
dc.contributor.institutionUniversity of St Andrews.Biomedical Sciences Research Complexen
dc.contributor.institutionUniversity of St Andrews.Centre of Magnetic Resonanceen
dc.identifier.doihttps://doi.org/10.1016/j.bpj.2017.09.005
dc.description.statusPeer revieweden


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