ABSTRACT
ß-Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well-defined secondary structures. Herein, we present the synthesis of the paramagnetic ß-amino acid ß3 -hTOPP (4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-d-ß3 -homophenylglycine) that enables investigations of ß-peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of ß-peptide spin labels. Its performance was evaluated by investigating the helical turn of a 314 -helical transmembrane model ß-peptide. Nanometer distances between two incorporated ß3 -hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi-rigid conformational design, the label delivers reliable distances and sharp (one-peak) distance distributions even in the lipid bilayer. The results indicate that the investigated ß-peptide folds into a 3.2514 helix and maintains this conformation in the lipid bilayer.
Subject(s)
Lipid Bilayers , Peptides , Electron Spin Resonance Spectroscopy , Lipid Bilayers/chemistry , Nitrogen Oxides/chemical synthesis , Nitrogen Oxides/chemistry , Peptides/chemistry , Protein Structure, Secondary , Spin Labels/chemical synthesisABSTRACT
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.
Subject(s)
Cell Membrane/chemistry , Cyclic N-Oxides/chemistry , Diketopiperazines/chemistry , Glycine/analogs & derivatives , Lipid Bilayers/chemistry , Peptides/chemistry , Amino Acid Sequence , Electron Spin Resonance Spectroscopy , Glycine/chemistry , Models, Molecular , Protein Conformation, alpha-Helical , Spin LabelsABSTRACT
Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson-Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the "ideal" A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an "ideal" A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.