Abstract

Pulse electron paramagnetic resonance (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., homomultimers) due to distorting multispin 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 multispin problem. Here, two different approaches for suppressing multispin 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 Escherichia 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 multispin 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 analog.

Highlights

  • Pulse electron paramagnetic resonance (EPR) has become an important tool in structural biology

  • Compared to more established methods such as x-ray crystallography, Forster resonance energy transfer, or nuclear magnetic resonance spectroscopy, pulse EPR spectroscopy is not dependent on the growth of crystals, with measurements performed in frozen aqueous solutions; the pulsed electron-electron double resonance (PELDOR) technique does not require the presence of different labels, and is not limited by rotational correlation times or the complexity of the system investigated

  • Distance distributions predicted by MMM and MtsslWizard were in good agreement with each other

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Summary

Introduction

Pulse electron paramagnetic resonance (EPR) has become an important tool in structural biology. PELDOR is highly complementary to other biophysical techniques employed in structural biology, most importantly offering the opportunity to accurately measure distances in the nanometer range The dipolar interaction, recorded as an oscillation during the dipolar evolution, is processed to obtain the corresponding distance distribution This is most usually done using a mathematical procedure called ‘‘Tikhonov regularization’’ within the DeerAnalysis software developed to PELDOR on Sparsely Labeled MscS or MscL analyze PELDOR data [8]. In this context, Tikhonov regularization is employed to best stabilize the solution of the moderately ill-posed inverse problem of going from time domain to distance data

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