Spin Label Spin Label Distance Measurements of Aligned Membrane Proteins

Abstract

Both Manganin-2 and M2δ transmembrane segment of the nicotinic acetylcholine receptor (AchR M2δ) are 23-amino acid peptides with strikingly different structural topologies. Manganin-2 is an antimicrobial peptide lying on the surface, whereas AchR M2δ forms a membrane-spanning neurotransmitter gated ion-channel. To determine the structural topology of these peptides, the spin label 2, 2, 6, 6-tetramethyl piperidine-1 oxyl-4-amino-4 carboxylic acid (TOAC) was attached at different sites in these peptides via solid phase peptide synthesis. Since the TOAC spin label is rigidly coupled to the peptide backbone, it accurately reports on position, orientation, and dynamics of the peptide backbone. Amino acids Ser-8 and Lys-14 in Manganin-2 and Ile-7 and Gln-13 in AchR M2δ were singly and doubly replaced with the TOAC spin label. These peptides were cleaved, purified by reverse phase HPLC and then inserted into aligned DMPC lipid bilayers to study the spin label-spin label dipolar interaction by continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. In aligned bilayers at higher temperatures, the anisotropic EPR spectra of the singly labeled peptides reveal unique lineshapes based upon the orientation of the peptides with respect to the membrane and the magnetic field. The EPR spectra of doubly labeled peptides in aligned bilayers contain unique information about the distance and orientation of the two labels. Thus, by using aligned CW-EPR spectroscopic techniques we can predict the structural topology of membrane proteins and measure the distance and orientation between two spin labels.