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).
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 four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, 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 X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and 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.
Ghimire
Harishchandra
H
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
McCarrick
Robert M
RM
Budil
David E
DE
Lorigan
Gary A
GA
D020816:Amino Acid Motifs; D050976:Basic-Leucine Zipper Transcription Factors / Q000032:analysis / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D016350:Leucine Zippers; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry*; D019097:Spin Trapping / Q000379:methods
D050976:Basic-Leucine Zipper Transcription Factors; C501391:GCN4 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins