MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00723-012-0314-0) contains supplementary material, which is available to authorized users.

One of the major problems facing distance determination by pulsed EPR, on spin-labeled proteins, has been the short relaxation time T(m). Solvent deuteration has previously been used to slow relaxation and so extend the range of distance measurement and sensitivity. We demonstrate here that deuteration of the underlying protein, as well as the solvent, extends the T(m) to a considerable degree. Longer T(m) gives greatly enhanced sensitivity, much extended distance measurement, more reliable distance distribution calculation and better baseline correction.

The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spinlabeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.DEER | electron paramagenetic resonance | ion channels | dipolar coupling

SummaryHistone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

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