The alignment of membrane proteins provides pertinent structural and dynamic information. Structural topology data gleaned from such studies can be used to determine the functional mechanisms associated with a wide variety of integral membrane proteins. In this communication, we successfully demonstrate, for the first time, the determination of the structural topology and helical tilt of an antimicrobial peptide magainin 2 using aligned X-band spin-label EPR spectroscopic techniques. This novel comparison unlocks many possibilities utilizing EPR spectroscopy to probe antimicrobial peptide topologies with increased sensitivity and may also give further clues to elucidate their corresponding mechanisms.
Theoretical calculations of hyperfine splitting values derived from the EPR spectra of TOAC spin labeled rigid aligned α-helical membrane peptides reveal a unique periodic variation. In the absence of helical motion, a plot of the corresponding hyperfine splitting values as a function of residue number results in a sinusoidal curve that depends on the helical tilt angle that the peptide makes with respect to the magnetic field. Motion about the long helical axis reduces the amplitude of the curve and averages out the corresponding hyperfine splitting values. The corresponding spectra can be used to determine the director axis tilt angle from the TOAC spin label, which can be used to calculate the helical tilt angle due to the rigidity of the TOAC spin label. Additionally, this paper describes a method to experimentally determine this helical tilt angle from the hyperfine splitting values of three consecutive residues.
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.
Acetylcholine receptors (AChRs) mediate rapid synaptic transmission by transducing a chemical signal into an electrical impulse. Transduction comprises binding of agonist followed by opening of the AChR ion channel, and in the classical view both processes depend on the agonist. However previous studies suggest the ultimate channel opening step is agonist-independent 1,2 , and is preceded by a priming step facilitated by the agonist 3 . Here, by studying mutant AChRs, we detect two such priming steps; the first generates a closed state that elicits brief openings, and the second generates a closed state that elicits long-lived openings. Long-lived openings and the associated priming step are detected in the absence of agonist and in its presence, and show identical kinetics under each condition. By covalently locking the agonist binding sites in the bound conformation, we show that each site initiates a priming step. Thus a change in binding site conformation primes the AChR for channel opening in a process that determines the maximum response to agonist and functional consequences of disease-causing mutations.
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