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

Abstract

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5-10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes.

Keywords: DEER; Dipolar spectroscopy; PDS; PELDOR; Pulsed ESR; SDSL; Spin label; Transmembrane peptide; α-TOPP; β-TOPP; β-peptide.

Fig. 1

Illustration of possible scenarios of peptide adaptation within a membrane (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

Fig. 2

a Four-pulse DEER/PELDOR sequence. The pump pulse is shifted between the 2^nd and the 3^rd detection pulses to induce the modulation of the refocused electron spin echo if dipolar coupling exists. The integrated echo signal is recorded as a function of time t. The modulation frequency encodes the information on distances between the electron spins. Times τ₁ and τ₂ are delays between the pulses in the mw detection sequence. τ_d is a delay for the pumping pulse. b A typical normalized DEER/PELDOR trace and its modulation depth λ

Fig. 3

Molecular structures of selected spin labels for protein and peptide labelling. α-TOPP and β-TOPP represent novel semi-rigid labels developed for incorporation into TM peptide by the solid phase peptide synthesis

Fig. 4

Top: WALP24 peptide sequence. Labelling positions (X) are marked in red. Center: TOPP (left) and MTSSL (right) spin labels and their rotamer distributions as modelled in Ref. (Halbmair et al. 2016). Bottom: Q-band DEER/PELDOR time traces (dots) recorded on TOPP-WALP24 (left) and MTSSL-WALP24 (right) in MeOH and their fits (lines). Experimental and modeled distance distributions are shown in insets (filled grey lines and red areas, respectively). Figure adapted from Ref. (Halbmair et al. 2016)

Fig. 5

Top: Schematic representation of D31-POPC and D54-DMPC as compared to the length of WALP24. Bottom: Q-band DEER/PELDOR traces of TOPP (left) and MTSSL (right) labelled WALP24 in different environments. Distance distributions obtained using DeerAnalysis are shown in insets. Figure adapted from Ref. (Halbmair et al. 2016)

Fig. 6

Sequence of the investigated β-peptides and results of CD and DEER spectroscopy. a Positions of β-TOPP labels (R) within the peptide sequence for different peptides (P2-P5). b Upper row: Measurements in MeOH. Left: CD spectra; Center: Q-band DEER/PELDOR traces (black lines); Right: distance distributions derived with DeerAnalysis. Lower row: Measurements in SUVs of POPC. Left: CD spectra; Center: Q-band DEER/PELDOR traces (black lines) and their DeerAnalysis fits (colored lines); Right: distance distributions derived with DeerAnalysis (colored lines). Colors correspond to peptide color coding in (a). Figure adapted from Ref. (Wegner et al. 2019)

Fig. 7

a Models of the labelled β³-peptides produced according to 3.25₁₄ torsion angle set. b Inter-spin distances derived with 3.25₁₄ (turquoise) and 3.0₁₄ (green) backbone angle sets, and experimental peak distances in MeOH (black) and POPC (red) for comparison. The inter-spin distances are plotted against the number of amino acids separating the two spin labels in P2–P5. Figure adapted from Ref. (Wegner et al. 2019)

Fig. 8

Schematic structure (PyMol, DeLano Scientific LLC) of the α-helical peptide containing α-TOPP-labels (positions Y) employed for orientation studies, adapted from (Tkach et al. 2013). Magnetic g-tensors for nitroxides and the tensor transformations defining the mutual label orientation are illustrated. $D_{\Vert }$ and $D_{\perp }$ represent the parallel and perpendicular principal axis values of the dipolar tensor. $R\left({\psi }_1\right)$ and $R\left({\psi }_2\right)$ describe label librations also considered in the simulations. Inter-spin distance of 2.8 ± 0.2 was determined at 9 GHz

Fig. 9

94 GHz DEER/PELDOR of an Ala-rich α-TOPP labelled peptide at fixed a and variable b frequency separation. a Left: schematic tuning picture of a commercial, single mode resonator used for 94 GHz experiments at fixed frequency separation (Δν = 56 MHz) and selected pump and detection position in the EPR line. Center/Right: FT-dipolar spectra (Pake patterns) and time traces obtained with detection across the EPR line. b Left: Variable frequency approach with a dual-mode resonator, pump and detection positions are indicated on the EPR spectrum. Center/Right: FT-dipolar spectra and time traces obtained for variable frequency separations and indicated detection positions. Adapted from Tkach et al. (2013)

Fig. 10

a, b Examples of randomly and partially oriented peptide samples in membranes and expected complete and incomplete Pake patterns for both arrangements. The incomplete Pake patterns in B are representing the arrangements with the interconnecting vectors r parallel and perpendicular to the magnetic field B₀ (upper and lower patterns, respectively). c Experimental setup to measure peptide tilt angles in aligned lipids using PDS in combination with SDSL consisting of an aligned membrane on a plate, inserted into an EPR tube. A goniometer can be employed to systematically vary the angle α, from which the dipolar response is detected (Dzikovski et al. 2011)