Significantly Improved Sensitivity of Q-Band PELDOR/DEER Experiments Relative to X-Band Is Observed in Measuring the Intercoil Distance of a Leucine Zipper Motif Peptide (GCN4-LZ)

Abstract

Pulsed Electron Double Resonance (PELDOR)/Double Electron-Electron Resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the 4-pulse DEER sequence and can often require up to 12+ hours of signal averaging for biological samples (depending upon spin label concentration). In this rapid report, we present for the first time, a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to Xband. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents 169-fold decrease in data-collection time, reveals greatly improved frequency spectrum, higher quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.Pulsed Electron Double Resonance (PELDOR)/Double Electron-Electron Resonance (DEER) spectroscopy is a rapidly emerging, powerful structural biology technique in which the dipolar coupling between two unpaired electron spins (usually site-directed nitroxide spin labels) is measured(1-3). The strength of the dipolar coupling can then be used to determine the distance between the two spins in the range of 2-8 nm(4-9). This allows researchers to gain valuable structural information from samples in which other techniques like solution NMR or X-ray crystallography prove difficult or impossible(10-12). These measurements are typically conducted with X-band (9.4 GHz) microwave excitation using the 4-pulse DEER sequence and can often require up to 12+ hours of signal averaging (depending upon concentration) for typical biological samples. In this communication, we report for the first time a substantial increase in sensitivity, that is obtained by collecting DEER spectra using a pulse Q-band (34 GHz) EPR spectrometer. The huge boost in sensitivity at Q-band reveals higher quality data and significantly reduces data acquisition time.For this study, an α-helical coiled coil peptide with a Leucine Zipper (LZ) motif (residues 245-281) of the yeast transcriptional activator GCN4(13,14) (PDB entry 1YSA) was used as a model. The peptide was synthesized on a solid-state peptide synthesizer with a single TOAC nitroxide spin label at position 248 with Gln→TOAC substitution for the distance measurements between the two monomers. The TOAC(15-19) spin label was chosen over the traditional MTSL (1,14) to eliminate disproportionation of the two label disulfide bonds to * To whom correspondence is to be addressed: Gary A. Lorigan Email: garylorigan@muohio.edu, Tel: 513-529-3338, Fax: 513-529-5715. form a linked peptide dimer, and to avoid the motional flexibility of the MTSL nitroxide group for more accurate inter-coil backbone distance measurements. In previously published r...

DEER Distance Measurements on Proteins

“…The huge sensitivity increase at modest microwave (MW) power claimed in later work (35) cannot be reproduced in our hands and is most likely a result of not fully optimizing the X-band measurement. More realistic results were reported later (33,36), although the X-band data taken for comparison still suffer from three times longer pump pulses than would have been been possible with available MW power.…”

Distance measurements across randomly distributed nitroxide probes from the temperature dependence of the electron spin phase memory time at 240GHz

“…High field EPR has been driven by many motives, including interest in accessing faster time scales, increased spectral resolution and sensitivity, and the ability to study high-spin species with zero-field splittings too large to be observed at low fields [35]. Pulsed Dipolar Spectroscopy has been implemented at several fields above 0.35 T, which allowed demonstration of improvements in sensitivity [36,37], and offers the capability to perform orientation selective measurements [38][39][40][41].…”

Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy

“…The origin of this striking result lies at the convergence of the intrinsic increase in signal-to-noise achieved with higher microwave frequencies relative to X-band (9.5 GHz) and the reduced contribution of background intermolecular dipolar coupling in the Nanodisc environment (Ghimire, McCarrick, Budil, & Lorigan, 2009; Zou & McHaourab, 2010). In proteoliposomes, multiple proteins can incorporate into a single liposome, which compresses the bulk spatial distribution of spins from three dimensions to approximately two (Figure 11) (Hilger et al, 2005).…”