Time-resolved EPR immersion depth studies of a transmembrane peptide incorporated into bicelles

Abstract

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n = 5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n = 5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2δ AChR peptide, the depth of immersion was calculated to be 5.8 Å and 3 Å for Glu-1, and 21.7 Å and 19 Å for Ala-12 in the gel-phase (298 K) and Lα-phases (318 K), respectively. The immersion depth values are consistent with the pitch of an α–helix and the structural model of M2δ AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2δ AChR peptide.

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

“…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]. It was shown by EPR that α-synuclein in bicelles forms an extended α-helix rather than a helix-turn-helix structure [134].…”

Membrane Permeabilities of Ascorbic Acid and Ascorbate

“…If the calculation was correct, would appear inside lipid vesicles of diameter d = 100 nm after time [ 37 ] had elapsed subsequent to addition to the outer solution. Yet, both a recent time resolved electron paramagnetic resonance(EPR) immersion depth study [ 38 ] and the original EPR study [ 10 ] indicate that it takes 20–30 min to penetrate to a probe that is buried at a depth of 20 Å of a fluid lipid bilayer. The of would completely rule out the possibility of using ascorbate to monitor lipid flip-flop [ 39 ], the more so, the neutral species AscH would permeate 10 6 times faster (see Equation (11)).…”

Kinetics of peptide folding in lipid membranes

“…Specifically, the following FRET pairs have been used to investigate the folding kinetics and mechanisms of peptides or proteins in a membrane environment: naphthalene/dansyl, pyrene/dansyl, Phe CN /Trp, Trp/dansyl, Trp/DBO, and Trp/Laurdan . Besides fluorescence spectroscopy, other kinetics methods, including stopped‐flow circular dichroism (CD), hydrogen/deuterium (H/D) exchange, SDS‐PAGE, and time‐resolved electron paramagnetic resonance spectroscopy, are powerful tools for investigating the mechanism of membrane peptide and protein folding.…”