Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Crystal structure validation of verinurad via proton-detected ultra-fast MAS NMR and machine learning.

The recent development of ultra-fast magic-angle spinning (MAS) (>100 kHz) provides new opportunities for structural characterization in solids. Here, we use NMR crystallography to validate the structure of verinurad, a microcrystalline active pharmaceutical ingredient. To do this, we take advantage of 1H resolution improvement at ultra-fast MAS and use solely 1H-detected experiments and machine learning methods to assign all the experimental proton and carbon chemical shifts. This framework provides a new tool for elucidating chemical information from crystalline samples with limited sample volume and yields remarkably faster acquisition times compared to 13C-detected experiments, without the need to employ dynamic nuclear polarization.

The Active Site of a Prototypical "Rigid" Drug Target is Marked by Extensive Conformational Dynamics.

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off-target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly "rigid protein" that substantial active-site dynamics have generally remained unrecognized, despite thousands of medicinal-chemistry studies on this model over decades. Comparing cryogenic X-ray structures, solid-state NMR on micro-crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational-exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

Assessment of a Large Enzyme-Drug Complex by Proton-Detected Solid-State NMR Spectroscopy without Deuteration.

Solid-state NMR spectroscopy has recently enabled structural biology with small amounts of non-deuterated proteins, largely alleviating the classical sample production demands. Still, despite the benefits for sample preparation, successful and comprehensive characterization of complex spin systems in the few cases of higher-molecular-weight proteins has thus far relied on traditional 13 C-detected methodology or sample deuteration. Herein we show for a 29 kDa carbonic anhydrase:acetazolamide complex that different aspects of solid-state NMR assessment of a complex spin system can be successfully accessed using a non-deuterated, 500 µg sample in combination with adequate spectroscopic tools. The shown access to protein structure, protein dynamics, as well as biochemical parameters in amino acid sidechains, such as histidine protonation states, will be transferable to proteins that are not expressible in E. coli.

Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

A False-Positive Screening Hit in Fragment-Based Lead Discovery: Watch out for the Red Herring.

With the rising popularity of fragment-based approaches in drug development, more and more attention has to be devoted to the detection of false-positive screening results. In particular, the small size and low affinity of fragments drives screening techniques to their limit. The pursuit of a false-positive hit can cause significant loss of time and resources. Here, we present an instructive and intriguing investigation into the origin of misleading assay results for a fragment that emerged as the most potent binder for the aspartic protease endothiapepsin (EP) across multiple screening assays. This molecule shows its biological effect mainly after conversion into another entity through a reaction cascade that involves major rearrangements of its heterocyclic scaffold. The formed ligand binds EP through an induced-fit mechanism involving remarkable electrostatic interactions. Structural information in the initial screening proved to be crucial for the identification of this false-positive hit.

Sequential backbone assignment based on dipolar amide-to-amide correlation experiments.

Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common (13)C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR.

Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins.

Solid-state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic-resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long-distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton-proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin-labeled 1 mg protein samples using fast magic angle spinning.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Integrated Assessment of the Structure and Dynamics of Solid Proteins.

Understanding macromolecular function, interactions, and stability hinges on detailed assessment of conformational ensembles. For solid proteins, accurate elucidation of the spatial aspects of dynamics at physiological temperatures is limited by the qualitative character or low abundance of solid-state nuclear magnetic resonance internuclear distance information. Here, we demonstrate access to abundant proton-proton internuclear distances for integrated structural biology and chemistry with unprecedented accuracy. Apart from highest-resolution single-state structures, the exact distances enable molecular dynamics (MD) ensemble simulations orchestrated by a dense network of experimental interproton distance boundaries gathered in the context of their physical lattices. This direct embedding of experimental ensemble distances into MD will provide access to representative, atomic-level spatial details of conformational dynamics in supramolecular assemblies, crystalline and lipid-embedded proteins, and beyond.

Unambiguous Side-Chain Assignments for Solid-State NMR Structure Elucidation of Nondeuterated Proteins via a Combined 5D/4D Side-Chain-to-Backbone Experiment.

Owing to fast-magic-angle-spinning technology, proton-detected solid-state NMR has been facilitating the analysis of insoluble, crystalline, sedimented, and membrane proteins. However, potential applications have been largely restricted by limited access to side-chain resonances. The recent availability of spinning frequencies exceeding 100 kHz in principle now allows direct probing of all protons without the need for partial deuteration. This potentiates both the number of accessible target proteins and possibilities to exploit side-chain protons as reporters on distances and interactions. Their low dispersion, however, has severely compromised their chemical-shift assignment, which is a prerequisite for their use in downstream applications. Herein, we show that unambiguous correlations are obtained from 5D methodology by which the side-chain resonances are directly connected with the backbone. When further concatenated with simultaneous 4D intra-side-chain correlations, this yields comprehensive assignments in the side chains and hence allows a high density of distance restraints for high-resolution structure calculation from minimal amounts of protein.

Protein Motional Details Revealed by Complementary Structural Biology Techniques.

Proteins depend on defined molecular plasticity for their functionality. How to comprehensively capture dynamics correctly is of ubiquitous biological importance. Approaches commonly used to probe protein dynamics include model-free elucidation of site-specific motion by NMR relaxation, molecular dynamics (MD)-based approaches, and capturing the substates within a dynamic ensemble by recent eNOE-based multiple-structure approaches. Even though MD is sometimes combined with ensemble-averaged NMR restraints, these approaches have largely been developed and used individually. Owing to the different underlying concepts and practical requirements, it has remained unclear how they compare, and how they cross-validate and complement each other. Here, we extract and compare the differential information contents of MD simulations, NMR relaxation measurements, and eNOE-based multi-state structures for the SH3 domain of chicken α-spectrin. The data show that a validated, consistent, and detailed picture is feasible both for timescales and actual conformational states sampled in the dynamic ensemble. This includes the biologically important side-chain plasticity, for which experimentally cross-validated assessment is a significant challenge.

Exact distance measurements for structure and dynamics in solid proteins by fast-magic-angle-spinning NMR.

Fast-magic-angle-spinning solid-state NMR is a developing technique for determination of protein structure and dynamics. Proton-proton correlations usually lead to rough distance restraints, a serious hurdle towards high-resolution structures. Analogous to the "eNOE" concept in solution, an integrative approach for more accurate restraints enables improved structural accuracy with minimal analytical effort.