Probing topology and dynamics of the second transmembrane domain (M2δ) of the acetyl choline receptor using magnetically aligned lipid bilayers (bicelles) and EPR spectroscopy

Abstract

Characterizing membrane protein structure and dynamics in the lipid bilayer membrane is very important but experimentally challenging. EPR spectroscopy offers a unique set of techniques to investigate a membrane protein structure, dynamics, topology, and distance constraints in lipid bilayers. Previously our lab demonstrated the use of magnetically aligned phospholipid bilayers (bicelles) for probing topology and dynamics of the membrane peptide M2δ of the acetyl choline receptor (AchR) as a proof of concept. In this study, magnetically aligned phospholipid bilayers and rigid spin labels were further utilized to provide improved dynamic information and topology of M2δ peptide. Seven TOAC-labeled AchR M2δ peptides were synthesized to demonstrate the utility of a multi-labeling amino acid substitution alignment strategy. Our data revealed the helical tilts to be 11°, 17°, 9°, 17°, 16°, 11°, 9° ± 4° for residues I7TOAC, Q13TOAC, A14TOAC, V15TOAC, C16TOAC, L17TOAC, and L18TOAC, respectively. The average helical tilt of the M2δ peptide was determined to be ~13°. This study also revealed that the TOAC labels were attached to the M2δ peptide with different dynamics suggesting that the sites towards the C-terminal end are more rigid when compared to the sites towards the N-terminus. The dynamics of the TOAC labeled sites were more resolved in the aligned samples when compared to the randomly disordered samples. This study highlights the use of magnetically aligned lipid bilayer EPR technique to determine a more accurate helical tilt and more resolved local dynamics of AchR M2δ peptide.

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

“…(D) Membrane depth parameter (φ) as a function of Q1VSD residue position in POPC/POPG lipid-bilayered vesicles at 295 K. (Adapted from [84] with permission). SDSL CW-EPR spectroscopy at the X-band can also be used to study membrane topology of membrane proteins/peptides bound to aligned phospholipid bilayers [86][87][88][89][90].…”

“…Figure 6 shows the membrane depth parameter as a function of the active pinholin S 21 68 residue position in DMPC lipid-bilayered vesicles at room temperature and the proposed topology model of the S 21 68 in DMPC lipid bilayers [66]. SDSL CW-EPR spectroscopy at the X-band can also be used to study membrane topology of membrane proteins/peptides bound to aligned phospholipid bilayers [86][87][88][89][90].…”

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins

“…Studies using EPR spectroscopy, pulse, and CW with spin labeling have also used bicelles as a lipid mimetic to study the conformational dynamics of IMPs. Magnetically aligned bicelles were used to probe the topology and orientation of the second transmembrane domain (M2δ) of the acetylcholine receptor using spin labeling and CW EPR [ 174 ]. Further, the immersion depth of the spin-labeled M2δ peptide at different positions in bicelles was determined.…”

Site-Directed Spin Labeling EPR for Studying Membrane Proteins

“…The result of this study suggested that the EPR spectra of a bifunctional spin label can be used to accurately determine the orientation and rotational dynamics of an α -helical segment of an integral membrane protein in magnetically aligned bicelles. Recently, the Lorigan lab determined accurate helical tilt angle and dynamics of the AchR M2 δ peptide by utilizing the magnetically aligned bicelles EPR technique and multiple TOAC labeled peptide substitutions [ 71 ].…”