Enhancement of Electron Spin Echo Envelope Modulation Spectroscopic Methods to Investigate the Secondary Structure of Membrane Proteins

Abstract

This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a 2H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. 2H-SL distance distributions from the MD results indicated that 2H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the α-helical M2δ peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with 2H-labeled Leu d10 at position 10 (i) and nitroxide spin labels positioned 1, 2, 3 and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an α-helix (3.6 residues per turn). Strong 2H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The 2H modulation depth observed for 2H-labeled d10 Leu was significantly enhanced (x4) when compared to previous ESEEM measurements that used 2H-labeled d8 Val. Computational studies indicate that deuterium nuclei on the Leu sidechain are closer to the spin label when compared to Val. The enhancement of 2H modulation and the corresponding Fourier Transform (FT) peak intensity for 2H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different 2H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (~50 μM).

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

“…These results reveal a unique ESEEM pattern for an α -helix which is consistent with previous ESEEM results. 11–13 Despite the longer side chain with more flexibility of the Leu amino acid, ESEEM spectra still revealed a similar pattern for this α -helix. At the same time, the modulation depth in the time domain data and the FT peak intensity in the frequency domain data of i − 3 and i − 4 positions were comparable to previous results.…”

“…At the same time, the modulation depth in the time domain data and the FT peak intensity in the frequency domain data of i − 3 and i − 4 positions were comparable to previous results. 11 The high signal-to-noise ratio of 2 H-labeled d 10 -Leu makes it a very efficient side chain probe for this ESEEM technique.…”

“…A cross-term averaged Fourier transformation (FT) was performed to the resulting spectrum to generate the corresponding frequency domain with minimized dead time artifacts. 11,12 Maximum deuterium peaks intensities at 2.3 MHz were measured and recorded for further analysis.…”

“…Thus, deuterium modulation would not be detected in the ESEEM time domain data or in the frequency domain data. 11–13 …”

“…11,12 Since 2 H-labeled d 10 -Leu has been shown as a very efficient 2 H-labeled probe for this ESEEM approach, a more indepth understanding about its ESEEM pattern and variations at different positions could be extremely helpful for its future application in biological systems. 11 Here, we further explore the ESEEM signal pattern of an α -helix with 2 H-labeled d 10 -Leu residues and provide a library of valuable data for this 2 H-labeled probe for the first time. Multiple 2 H-labeled d 10 -Leu residues on AChR M2 δ peptides were mapped out on both sides with SDSL to provide a more detailed description of the ESEEM pattern.…”