Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

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

Conformational Basis for Asymmetric Seeding Barrier in Filaments of Three- and Four-Repeat Tau

*S Supporting Information ABSTRACT: Tau pathology in Alzheimer’s disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron−electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand−turn−β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies

Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity

Site-Specific Information on Membrane Protein Folding by Electron Spin Echo Envelope Modulation Spectroscopy

Compared to folding of soluble proteins folding of membrane proteins is complicated by the fact that it requires an amphiphilic environment. Few existing techniques can provide structurally resolved information on folding kinetics. For the major plant light harvesting complex LHCII, it is demonstrated that changes in water accessibility of a particular amino acid residue can be followed during folding by measuring the hyperfine interaction of spin labels with deuterium nuclei of heavy water. The incorporation of residue 196 into the hydrophobic core of adetergent micelle was investaged. The technique provides a time constant that is similar to the one found with fluorescence spectroscopy for the slower folding step of the whole protein and with electron paramagnetic resonance for change of the distance between residues 90 and 196. If applied to several residues, this technique should provide information on the sequence of events during membrane protein folding

Analysis of influenza A virus NS1 dimer interfaces in solution by pulse EPR distance measurements

H.L.T. was supported by the University of St Andrews; S.A.J. is supported by the BBSRC, U.K.; P.S.K. is supported by the Medical Research Council, U.K.; B.E.B. is grateful for an EaStCHEM Hirst Academic Fellowship by the School of Chemistry, St Andrews, and funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme [REA 334496]. This work was supported by the Wellcome Trust [099149/Z/12/Z].Pulsed electron-electron double resonance (PELDOR) is an electron paramagnetic resonance (EPR) spectroscopy technique for nanometer distance measurements between paramagnetic centres such as radicals. PELDOR has been recognized as a valuable tool to approach structural questions in biological systems. In this manuscript we demonstrate the value of distance measurements for differentiating competing structural models on the dimerization of the effector domain (ED) of the non-structural protein 1 (NS1) of the influenza A virus. Our results show NS1 to be well amenable to nanometer distance measurements by EPR, yielding high quality data. In combination with mutants perturbing protein dimerization and in silico prediction based on crystal structures we can exclude one of two potential dimerization interfaces. Furthermore, our results lead to a viable hypothesis of a NS1 ED:ED interface which is flexible through rotation around the vector interconnecting the two native cysteines. These results prove the high value of pulse EPR as a complementary method for structural biology.Publisher PDFPeer reviewe