NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples

Abstract

We characterize the effect of specifically bound biradicals on the NMR spectra of dihydrofolate reductase from E. coli. Dynamic nuclear polarization methods enhance the signal-to-noise of solid state NMR experiments by transferring polarization from unpaired electrons of biradicals to nuclei. There has been recent interest in colocalizing the paramagnetic polarizing agents with the analyte of interest through covalent or noncovalent specific interactions. This experimental approach broadens the scope of dynamic nuclear polarization methods by offering the possibility of selective signal enhancements and the potential to work in a broad range of environments. Paramagnetic compounds can have other effects on the NMR spectroscopy of nearby nuclei, including broadening of nuclear resonances due to the proximity of the paramagnetic agent. Understanding the distance dependence of these interactions is important for the success of the technique. Here we explore paramagnetic signal quenching due to a bound biradical, specifically a biradical-derivatized trimethoprim ligand of E. coli dihydrofolate reductase. Biradical-derivatized trimethoprim has nanomolar affinity for its target, and affords strong and selective signal enhancements in dynamic nuclear polarization experiments. In this work, we show that, although the trimethoprim fragment is well ordered, the biradical (TOTAPOL) moiety is disordered when bound to the protein. The distance dependence in bleaching of NMR signal intensity allows us to detect numerous NMR signals in the protein. We present the possibility that static disorder and electron spin diffusion play roles in this observation, among other contributions. The fact that the majority of signals are observed strengthens the case for the use of high affinity or covalent radicals in dynamic nuclear polarization solid state NMR enhancement.

Blind spheres of paramagnetic dopants in solid state NMR

“…If a spherical regime around a paramagnetic centre is assumed, from which no NMR signals can be detected, it is possible to relate the signal loss to the size of this sphere of influence which is known under the name blind-sphere 38,39 or wipe-out radius. [40][41][42] Such radii are important in solution NMR 38,39 where paramagnetic constraints are used for structure solution, and in dynamic nuclear polarization (DNP) to estimate the zone which cannot be accessed by NMR [43][44][45] and for luminescent materials to study doping homogeneity. 21 Spectroscopically the blind sphere can be explained by line broadening which leads to undetectable signal by standard experiments, 39 or signal shift 46 that puts the signal outside of spectral window.…”

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites

“… 20 – 22 Recently several groups have discussed the use of a localized PA, either covalently linked to the system of interest or to a bound-ligand, or just using a PA with high-affinity for the system of interest. 21 , 23 – 29 The motivations behind these contributions were different from those for the work presented here, ranging from optimizing sample sensitivity through PA co-localization, 21 , 23 – 26 along with a reduction of the cryo-protectant content, 23 , 24 to the development of targeted DNP experiments in complex environments 27 or the experimental investigation of the bleaching effect using specific isotopic labelled samples. 28 , 29 …”

“… 21 , 23 – 29 The motivations behind these contributions were different from those for the work presented here, ranging from optimizing sample sensitivity through PA co-localization, 21 , 23 – 26 along with a reduction of the cryo-protectant content, 23 , 24 to the development of targeted DNP experiments in complex environments 27 or the experimental investigation of the bleaching effect using specific isotopic labelled samples. 28 , 29 …”

“…According to our DQ–SQ 2D data, this effect is noticeable up to at least 30 Å from the paramagnetic center, in agreement with DNP bleaching radii reported by other groups. 28 , 29 When the PA is uniformly distributed in the sample, partial broadening and signal attenuation statistically affect all resonances.…”

Targeted DNP for biomolecular solid-state NMR

“…Making use of spin labels in the context of MAS-DNP was thus a natural next step in the development of the technique, and several groups in the last years have investigated the benefits of tagging their biomolecular system with the PA required for hyperpolarization. 57,58 The motivation and objectives of the different approaches were diverse, going from optimization of the sample preparation for best sensitivity 59,60 and biocompatibility, [61][62][63] investigation of the bleaching/quenching effect of the paramagnetic tag [64][65][66] under DNP conditions for its potential use as a structural probe, [67][68][69] selection of a particular protein in a complex environment, 62,63,[70][71][72][73][74][75] to the detection of protein-protein interactions, 76 and the development of highresolution localized techniques through selective DNP. 77…”