Tools shaping drug discovery and development.

Spectroscopic, scattering, and imaging methods play an important role in advancing the study of pharmaceutical and biopharmaceutical therapies. The tools more familiar to scientists within industry and beyond, such as nuclear magnetic resonance and fluorescence spectroscopy, serve two functions: as simple high-throughput techniques for identification and purity analysis, and as potential tools for measuring dynamics and structures of complex biological systems, from proteins and nucleic acids to membranes and nanoparticle delivery systems. With the expansion of commercial small-angle x-ray scattering instruments into the laboratory setting and the accessibility of industrial researchers to small-angle neutron scattering facilities, scattering methods are now used more frequently in the industrial research setting, and probe-less time-resolved small-angle scattering experiments are now able to be conducted to truly probe the mechanism of reactions and the location of individual components in complex model or biological systems. The availability of atomic force microscopes in the past several decades enables measurements that are, in some ways, complementary to the spectroscopic techniques, and wholly orthogonal in others, such as those related to nanomechanics. As therapies have advanced from small molecules to protein biologics and now messenger RNA vaccines, the depth of biophysical knowledge must continue to serve in drug discovery and development to ensure quality of the drug, and the characterization toolbox must be opened up to adapt traditional spectroscopic methods and adopt new techniques for unraveling the complexities of the new modalities. The overview of the biophysical methods in this review is meant to showcase the uses of multiple techniques for different modalities and present recent applications for tackling particularly challenging situations in drug development that can be solved with the aid of fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, atomic force microscopy, and small-angle scattering.

Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy.

The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b5 complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional 13C/13C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b5. The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register.

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a ß-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems.

Efficient cross-effect dynamic nuclear polarization without depolarization in high-resolution MAS NMR.

Dynamic nuclear polarization (DNP) has the potential to enhance the sensitivity of magic-angle spinning (MAS) NMR by many orders of magnitude and therefore to revolutionize atomic resolution structural analysis. Currently, the most widely used approach to DNP for studies of chemical, material, and biological systems involves the cross-effect (CE) mechanism, which relies on biradicals as polarizing agents. However, at high magnetic fields (≥5 T), the best biradicals used for CE MAS-DNP are still far from optimal, primarily because of the nuclear depolarization effects they induce. In the presence of bisnitroxide biradicals, magic-angle rotation results in a reverse CE that can deplete the initial proton Boltzmann polarization by more than a factor of 2. In this paper we show that these depolarization losses can be avoided by using a polarizing agent composed of a narrow-line trityl radical tethered to a broad-line TEMPO. Consequently, we show that a biocompatible trityl-nitroxide biradical, TEMTriPol-1, provides the highest MAS NMR sensitivity at ≥10 T, and its relative efficiency increases with the magnetic field strength. We use numerical simulations to explain the absence of depolarization for TEMTriPol-1 and its high efficiency, paving the way for the next generation of polarizing agents for DNP. We demonstrate the superior sensitivity enhancement using TEMTriPol-1 by recording the first solid-state 2D 13C-13C correlation spectrum at natural isotopic abundance at a magnetic field of 18.8 T.

Gd(iii) and Mn(ii) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins.

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = -1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(iii) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies.

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals.

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization.

While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from ¹³C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement. Further, results obtained from a 2D ¹³C/¹³C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

A method for dynamic nuclear polarization enhancement of membrane proteins.

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.

Selective host-guest interaction between metal ions and metal-organic frameworks using dynamic nuclear polarization enhanced solid-state NMR spectroscopy.

The host-guest interaction between metal ions (Pt(2+) and Cu(2+) ) and a zirconium metal-organic framework (UiO-66-NH2 ) was explored using dynamic nuclear polarization-enhanced (15) N{(1) H} CPMAS NMR spectroscopy supported by X-ray absorption spectroscopy and density functional calculations. The combined experimental results conclude that each Pt(2+) coordinates with two NH2 groups from the MOF and two Cl(-) from the metal precursor, whereas Cu(2+) do not form chemical bonds with the NH2 groups of the MOF framework. Density functional calculations reveal that Pt(2+) prefers a square-planar structure with the four ligands and resides in the octahedral cage of the MOF in either cis or trans configurations.

Dynamic nuclear polarization NMR enables the analysis of Sn-Beta zeolite prepared with natural abundance ¹¹9Sn precursors.

The catalytic activity of tin-containing zeolites, such as Sn-Beta, is critically dependent on the successful incorporation of the tin metal center into the zeolite framework. However, synchrotron-based techniques or solid-state nuclear magnetic resonance (ssNMR) of samples enriched with (119)Sn isotopes are the only reliable methods to verify framework incorporation. This work demonstrates, for the first time, the use of dynamic nuclear polarization (DNP) NMR for characterizing zeolites containing ~2 wt % of natural abundance Sn without the need for (119)Sn isotopic enrichment. The biradicals TOTAPOL, bTbK, bCTbK, and SPIROPOL functioned effectively as polarizing sources, and the solvent enabled proper transfer of spin polarization from the radical's unpaired electrons to the target nuclei. Using bCTbK led to an enhancement (ε) of 75, allowing the characterization of natural-abundance (119)Sn-Beta with excellent signal-to-noise ratios in <24 h. Without DNP, no (119)Sn resonances were detected after 10 days of continuous analysis.

Higher order amyloid fibril structure by MAS NMR and DNP spectroscopy.

Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure, and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105-115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide ß-strands into ß-sheets but also the ß-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The ß-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the residues buried within the interior of the fibrils.

Shortening spin-lattice relaxation using a copper-chelated lipid at low-temperatures - A magic angle spinning solid-state NMR study on a membrane-bound protein.

Inherent low sensitivity of NMR spectroscopy has been a major disadvantage, especially to study biomolecules like membrane proteins. Recent studies have successfully demonstrated the advantages of performing solid-state NMR experiments at very low and ultralow temperatures to enhance the sensitivity. However, the long spin-lattice relaxation time, T1, at very low temperatures is a major limitation. To overcome this difficulty, we demonstrate the use of a copper-chelated lipid for magic angle spinning solid-state NMR measurements on cytochrome-b5 reconstituted in multilamellar vesicles. Our results on multilamellar vesicles containing as small as 0.5mol% of a copper-chelated lipid can significantly shorten T1 of protons, which can be used to considerably reduce the data collection time or to enhance the signal-to-noise ratio. We also monitored the effect of slow cooling on the resolution and sensitivity of (13)C and (15)N signals from the protein and (13)C signals from lipids.

Dynamic nuclear polarization NMR spectroscopy allows high-throughput characterization of microporous organic polymers.

Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance (13)C and (15)N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods.

Sensitivity-enhanced solid-state NMR detection of expansin's target in plant cell walls.

Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of (13)C spin diffusion from trace concentrations of (13)C, (15)N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short (13)C-(13)C distances of 4-6 Å between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D (13)C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets.

Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology.

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear (13)C and (15)N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.

Atomic structure and hierarchical assembly of a cross-ß amyloid fibril.

The cross-ß amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of ß-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-ß amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent ß-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Uniform broadband excitation of crystallites in rotating solids using interleaved sequences of delays alternating with nutation.

In solids that are spinning about the magic angle, trains of short pulses in the manner of Delays Alternating with Nutations for Tailored Excitation (DANTE) allow one to improve the efficiency of the excitation of magnetization compared to rectangular pulses. By interleaving N pulse trains with N>1, one obtains 'DANTE-N' sequences comprising N pulses per rotor period that extend over K rotor periods. Optimized interleaved DANTE schemes with N>1 are shorter than basic DANTE-1 sequences with N=1. Therefore, they are less affected by coherent or incoherent decays, thus leading to higher signal intensities than can be obtained with basic DANTE-1 or with rectangular pulses. Furthermore, the shorter length of DANTE-N with N>1 increases the width of the spikelets in the excitation profile, allowing one to cover the range of isotropic chemical shifts and second-order quadrupolar effects typical for side-chain and backbone amide (14)N sites in peptides at B(0)=18.8 T. In DANTE-N, spinning sidebands only appear at multiples of the spinning frequency ν(rot), as if the samples were rotating at Nν(rot). We show applications to direct detection of nitrogen-14 nuclei with spin I=1 subject to large quadrupole interactions, using fast magic angle spinning (typically ν(rot)≥60 kHz), backed up by simulations that provide insight into the properties of basic and interleaved DANTE sequences. When used for indirect detection, we show by numerical simulations that even basic DANTE-1 sequences can lead to a four-fold boost of efficiency compared to standard rectangular pulses.

Dynamic nuclear polarization of quadrupolar nuclei using cross polarization from protons: surface-enhanced aluminium-27 NMR.

The surface of γ-alumina nanoparticles can be characterized by dynamic nuclear polarization (DNP) surface-enhanced NMR of (27)Al. DNP is combined with cross-polarization and MQ-MAS to determine local symmetries of (27)Al sites at the surface.