Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The CuII-Trityl Case.

Recent sensitivity enhancements in pulse dipolar electron paramagnetic resonance spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin-labeling using CuII ions and trityl yields a >3-fold increase in sensitivity compared to that of the established CuII-nitroxide labeling strategy. Application of the recently developed variable-time relaxation-induced dipolar modulation enhancement (RIDME) method yields a further ∼2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, >2 times lower than the previously reported concentration. We expect that experiments at single-digit nanomolar concentrations are imminent, which have the potential to transform biological PDS applications.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (NTA) Motif.

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu2+ (NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron-electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu2+ (NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+ (NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu2+ (NTA) motif in PDS experiments.

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

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.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

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.

SLIM: A Short-Linked, Highly Redox-Stable Trityl Label for High-Sensitivity In-Cell EPR Distance Measurements.

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in-cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide-functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.

Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy.

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology.

The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.