Pulse EPR Measurements of Intramolecular Distances in a TOPP-Labeled Transmembrane Peptide in Lipids

Abstract

We present the performance of nanometer-range pulse electron paramagnetic resonance distance measurements (pulsed electron-electron double resonance/double electron-electron resonance, PELDOR/DEER) on a transmembrane WALP24 peptide labeled with the semirigid unnatural amino acid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP). Distances reported by the TOPP label are compared to the ones reported by the more standard MTSSL spin label, commonly employed in protein studies. Using high-power pulse electron paramagnetic resonance spectroscopy at Q-band frequencies (34 GHz), we show that in contrast to MTSSL, our label reports one-peak, sharp (Δr ≤ 0.4 nm) intramolecular distances. Orientational selectivity is not observed. When spin-labeled WALP24 was inserted in two representative lipid bilayers with different bilayer thickness, i.e., DMPC and POPC, the intramolecular distance reported by TOPP did not change with the bilayer environment. In contrast, the distance measured with MTSSL was strongly affected by the hydrophobic thickness of the lipid. The results demonstrate that the TOPP label is well suited to study the intrinsic structure of peptides immersed in lipids.

Semi‐Rigid Nitroxide Spin Label for Long‐Range EPR Distance Measurements of Lipid Bilayer Embedded β‐Peptides

“…However, MTSSL is highly flexible, which leads to broad distance distributions especially in a lipid bilayer . We have recently reported that distances measured with MTSSL in the lipid bilayer can be affected by interactions of the label within the lipid environment . Therefore, interpretation of observed distances based on the MTSSL label is often challenging.…”

“…Therefore, interpretation of observed distances based on the MTSSL label is often challenging. For α‐peptides, we introduced the semi‐rigid nitroxide label 4‐(3,3,5,5‐tetramethyl‐2,6‐dioxo‐4‐oxylpiperazin‐1‐yl)‐ d ‐phenylglycine (TOPP) that enables structural investigation of transmembrane α‐peptides in their natural environment . Despite its sterical demand, the label did not show influence on the respective secondary structure and gave sharp distance distributions.…”

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

“…However, MTSSL is highly flexible, which leads to complex distance distributions especially in (Holt and Killian 2010) which can be studied using PDS and SDSL. a Tilting of the helix, b bending of the backbone, c stretching of the lipid acyl chains, d peptide aggregation lipid media (Halbmair et al 2016). Thus, an interpretation of the observed distances delivered by the label is often complicated.…”

In silico method for selecting residue pairs for single-molecule microscopy and spectroscopy

“…Double electron–electron resonance (DEER) or Pulsed Electron–electron double resonance (PELDOR) can be used to probe distance changes upon changing conditions 5 and single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain single molecule dynamics of proteins and other macromolecular assemblies 6 – 10 . The latter two techniques make use of two labels that are introduced for instance by attaching them to cysteine residues via maleimide 11 or methanethiosulfonate chemistry 12 or introducing them as non-natural amino acids 13 . SmFRET even enables study of protein dynamics in vivo 12 .…”