Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules

Abstract

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Figure 1.

Four-pulse PELDOR/DEER experiment. (a) The DEER/PELDOR pulse sequence with rectangular pulses of the same length. (b) Frequency-domain Q-band (~34 GHz) spectrum of a nitroxide spin label (black) along with the excitation profiles of 16 ns pump and probe pulses (in red and blue, respectively). The pump pulse is set to the maximum of the nitroxide spectrum and the probe pulse is set in this case 100 MHz lower in frequency. The excitation bandwidths are calculated for pulse lengths of 16 ns for both pump and probe pulses. (c) Background-corrected Q-band time trace with the modulation depth $\Delta$ indicated. (d) Schematic representation of two spins (red and blue spheres) connected via a distance vector $r$. $\theta$ is the angle between this distance vector and the applied magnetic field $B_0$.

Figure 2.

Structure of YopO and the spin labeling positions. (a–d) The rotamer clouds (mtsslWizard) of the R1 pairs are depicted in the same color code for the constructs: S353R1/Q635R1, cyan; V599R1/N624R1, orange; S585R1/Q603R1, green; and Y588R1/N624R1, pink. The distance within the pairs is indicated with a black dashed arrow. (a) Crystal structure of YopO_89–729 shown without the bound actin (PDB-ID: 4ci6). The kinase domain is shown in gray and the GDI domain in blue. (b) The same structure as in (a) but rotated by 120°. (c) Crystal structure of the isolated GDI domain (PDB-ID: 2h7o). Note, the GDI domain does not include position S353. (d) The same structure as in (c) but rotated by 120°. (e) Labeling reaction of MTSL with a cysteine giving the spin-labeled residue R1. The leaving group methanesulfinic acid reacts further with oxygen to methanesulfonic acid. The rotatable bonds of R1 are indicated by curly arrows.

Figure 3.

Q-band PELDOR/DEER time traces. Time traces for (a) S585R1/Q603R1, (b) V599R1/N624R1, (c) Y588R1/N624R1, and (d) S353R1/Q635R1. The time traces are color-coded according to the laboratories A–G. The time traces are shifted relative to each other for better visibility. The background fits done in DeerAnalysis (Versions 2015–2019) are overlaid as gray dotted lines.

Figure 4.

Overlays of the normalized distance distributions as obtained by Tikhonov regularization with DeerAnalysis. The distance distributions are color-coded according to laboratories A-G that processed them and uncertainty bands from DeerAnalysis validation are indicated as transparent areas: (a) S585R1/Q603R1, (b) V599R1/N624R1, (c) Y588R1/N624R1, and (d) S353R1/Q635R1.

Figure 5.

Schematic representation of the most common determinants of the width of the MTSL-derived distance distributions in PELDOR/DEER. (a) The five potentially rotatable bonds enable distinct rotamers to be populated at a specific site, based on the steric hindrance imposed by neighboring side chains and backbone atoms. Three rotamers are shown with arrows highlighting the rotatable bonds in one of them. (b) Small translational or rotational motion of the backbone to which the rotamers are attached can also induce broadening or appearance of shoulders in the distance distribution toward another spin-labeled site. (c) The protein adopts two distinct conformations (for example with and without a ligand bound) which can be monitored by PELDOR/DEER. Equilibria between two conformations can also be identified by the appearance of two peaks in the distance distribution. (d) If MTSL is attached to intrinsically disordered proteins or to a dynamic region of a protein, a broad distribution of distances is expected in frozen state. Such disorder is correlated with the large-amplitude motions of the backbone to which MTSL is attached.

Figure 6.

Classification of distance distributions encountered with MTSL-labeled proteins described in detail in the text. The main characteristics are related to the full width at half-maximum (FWHM) of the distance peaks, and by the presence of distance peaks separated by $\Delta r$. The most important aspects to be considered in interpretation are briefly summarized below each distance distribution.