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.

Dynamic Nuclear Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Combined with Molecular Dynamics Simulations Permits Detection of Order and Disorder in Viral Assemblies.

We report dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their life cycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic-level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking.

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix.

Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b 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.

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 β-helical arrangement, whereas others suggest a parallel in-register organization.

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 Gd and Mn to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of H, C, and N at magnetic fields of 5, 9.4, and 14.1 T.

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.

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.

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 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.

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.

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.

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.

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)).

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.

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.

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).

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.

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

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-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-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis.

Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A.

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore.

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.

Surface functionalization of alumina ceramic foams with organic ligands.

Different anchoring groups have been studied with the aim of covalently binding organic linkers to the surface of alumina ceramic foams. The results suggested that a higher degree of functionalization was achieved with a pyrogallol derivative--as compared to its catechol analogue--based on the XPS analysis of the ceramic surface. The conjugation of organic ligands to the surface of these alumina materials was corroborated by DNP-MAS NMR measurements.

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.