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

Abstract

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing 2H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was 2H-labeled, such as 2H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with 2H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique 2H-ESEEM patterns were observed with the 2H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the 2H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, 2H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.

Site-Directed Spin Labeling EPR for Studying Membrane Proteins

“…It has been used to investigate penetration of water into membranes, localization of proteins, or lipids in lipid membranes [ 77 – 81 ]. Recently SDSL ESEEM spectroscopy has been utilized to directly probe the local secondary structure of membrane proteins/peptides in aqueous as well as lipid membrane environments [ 52 , 79 , 80 , 82 – 86 ]. In this method, a cysteine mutated nitroxide spin label is positioned 2 ( i + 2), 3 ( i + 3), or 4 ( i + 4) residues away from a fully deuterated Leu side-chain ( i ).…”

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

“…Despite their physiological importance, minimal structural information is currently available as a result of limited biophysical techniques for studying these challenging protein systems [2,3]. More than 70% of membrane proteins with solved 3-D structures contain α-helical secondary structural motifs, which play important roles in assembly, packing, and membrane protein interactions [4]. Several established spectroscopic methods for studying secondary structural motifs like these include circular dichroism (CD), solid-state nuclear magnetic resonance (ss-NMR) spectroscopy, FT-IR, and FT-Raman [5–10].…”

“…The Lorigan lab has established a powerful electron spin echo envelope modulation (ESEEM) approach coupled with site-directed spin labeling (SDSL) to probe the local secondary structure of membrane proteins that is advantageous compared to other structural biology approaches because of the high sensitivity and short data acquisition times required [1,4]. ESEEM spectroscopy can detect weakly-coupled NMR active nuclei to nearby unpaired electron spins.…”

“…Distinct patterns are observed from the ESEEM data because of the unique turn periodicities of various secondary structural components, which provide insights into the type of secondary structures present in membrane proteins. Previously, we utilized this pulsed EPR method to identify site-specific secondary structural motifs such as α-helices [1,4,11], β-sheets [12], and more recently, 310-helices [13]. Site-specific secondary structural information provides a better understanding of the functions, dynamics, and protein-lipid interactions of membrane proteins [4,14].…”

“…Previously, we utilized this pulsed EPR method to identify site-specific secondary structural motifs such as α-helices [1,4,11], β-sheets [12], and more recently, 310-helices [13]. Site-specific secondary structural information provides a better understanding of the functions, dynamics, and protein-lipid interactions of membrane proteins [4,14]. Not only has this ESEEM approach been successful for model peptides, it has also been utilized in over-expression systems to probe α-helical secondary structural motifs in both a water-soluble protein and a membrane protein, demonstrating the versatility of this technique [15,16].…”