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.

Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield T_M relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.

Mechanistic insights into protein–ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA‐guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand‐induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site‐directed spin labeling (SDSL) with distance measurements based on pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose‐substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation–association mechanism for the formation of the twisted dimer upon ligand binding.

Pulsed electron paramagnetic resonance dipolar spectroscopy (PDS) allows to measure the distances between electron spin centers and, in favorable cases, their relative orientation. This data is frequently used in structural biology for studying biomolecular structures, following their conformational changes and localizing paramagnetic centers within them. In order to extract the inter-spin distances and the relative orientation of spin centers from the primary, time-domain PDS signals, a specialized data analysis is required. So far, the software to do such analysis was available only for isotropicS = 1/2 spin centers, such as nitroxide and trityl radicals, as well as for high-spin Gd³⁺and Mn²⁺ions. Here, a new data analysis program, called AnisoDipFit, was introduced for spin systems consisting of one isotropic and one anisotropicS = 1/2 spin centers. The program was successfully tested on the PDS data corresponding to the spin systems Cu²⁺/organic radical, low-spin Fe³⁺/organic radical, and high-spin Fe³⁺/organic radical. For all tested spin systems, AnisoDipFit allowed determining the inter-spin distance distribution with a sub-angstrom precision. In addition, the spatial orientation of the inter-spin vector with respect to theg-frame of the metal center was determined for the last two spin systems. Thus, this study expands the arsenal of the PDS data analysis programs and facilitates the PDS-based distance and angle measurements on the highly relevant class of metolloproteins.

Mechanistische Erkenntnisse über Protein‐Ligand‐Wechselwirkungen können chemische Werkzeuge für die Modulation von Proteinfunktionen liefern und deren Einsatz zu therapeutischen Zwecken ermöglichen. Für das homodimere Enzym tRNA‐Guanin‐Transglycosylase (TGT), eine virulenzrelevante Zielstruktur der Shigellose, wurde kristallographisch gezeigt, dass die Bindung bestimmter Liganden die funktionale Dimergeometrie in eine katalytisch‐inkompetente verdrehte Geometrie umwandelt. Die kristallographische Beobachtung beider Endzustände beweist jedoch weder die ligandeninduzierte Umwandlung zwischen beiden Dimerformen in Lösung noch gibt sie Aufschluss über den zugrunde liegenden Umwandlungsmechanismus. Wir sind diesen Fragen mit einem Ansatz nachgegangen, der ortsspezifische Spinmarkierung (SDSL) mit Abstandsmessungen auf der Grundlage der gepulsten Elektron‐Elektron‐Doppelresonanz‐Spektroskopie (PELDOR oder DEER) kombiniert. Wir beobachteten ein Gleichgewicht zwischen dem funktionalen und dem verdrehten Dimer, das von der chemischen Struktur des Liganden abhängt, wobei ein Pyranose‐substituierter Ligand das Gleichgewicht am stärksten in Richtung des verdrehten Dimers verschiebt. Unsere Experimente deuten auf einen Dissoziations‐Assoziations‐Mechanismus für die Bildung des verdrehten Dimers bei Ligandenbindung hin.

Robust external manipulation of spin communication in molecular frameworks is desirable for potential applications in quantum information science. Herein, we incorporate two types of dithienylethene (DTE) units into tris(2,4,6‐trichlorophenyl)methyl (TTM)‐based diradicaloids and report on the successful bistable switching of the optical and magnetic properties of the molecule. Visible‐light irradiation of the open form generates up to 97% of the closed form, in which conjugation between the two radical centers leads to absorption over the entire visible range of the spectrum as well as a twofold increase of the singlet–triplet gap. These results establish TTM/DTE hybrids as persistent and multi‐level responsive diradicaloids.