Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy

Abstract

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the a-helical M2d subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i 1 1 to i 1 4). The characteristic periodicity of the a-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i 1 1 and i 1 2 samples, the 2

Probing the Secondary Structure of Membrane Peptides Using 2H-Labeled d10-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy

“…11,12 2 H modulation can be detected for i ± 3 and i ± 4 positions, but not i ± 1 or i ± 2 positions. Beside the similar pattern that 2 H-labeled d 10 -Leu and 2 H-labeled d 8 -Val share for an α -helix, Leu demonstrated some unique features due to the longer and more flexible side chain.…”

“…A nitroxide spin label was attached to a mutated cysteine residue on a subsequent position on each sample (denoted as i + 1 to i + 4, yellow in Figure 1) which is one, two, three, or four amino acids away from the 2 H-labeled Leu. 12 ESEEM spectroscopy can detect the weak dipolar coupling between the spin label and 2 H atoms up to 8 Å. When the 2 H-labeled amino acid and spin-labeled cysteine are three or four amino acids away ( i + 3 or i + 4), the 2 H-labeled amino acid and the spin label point to the same side of the helix (Figure 1A).…”

When detergent meets bilayer: Birth and coming of age of lipid bicelles

“…The analysis method, which is similar to the dipolar waves [130] used in solid-state NMR, is described in detail elsewhere [131]. Similar results could be obtained in unoriented samples [132]. The quenching of EPR spin labels by water-soluble reducing agents can be monitored in real-time to determine details of membrane immersion, as demonstrated for the M2δ peptide [133].…”

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

“…By using SDSL coupled with ESEEM spectroscopy, the secondary structure of membrane peptides and proteins can be determined by detecting 2 H modulation between a 2 H-labeled amino acid and a nearby spin-labeled cysteine residue (Liu et al, 2012; Mayo et al, 2011; Zhou et al, 2012). A cysteine-mutated nitroxide spin label (MTSL) is positioned strategically at 1, 2, 3, and 4 residues away from an amino acid (i) with a deuterated side chain (denoted as i+1 to i+4).…”