An Efficient and Stable Polarizing Agent for In-Cell Magic-Angle Spinning Dynamic Nuclear Polarization NMR Spectroscopy.

Nuclear Magnetic Resonance (NMR) spectroscopy would be a method of choice to follow biochemical events in cells because it can analyze molecules in complex environments. However, the intrinsically low sensitivity of NMR makes in-cell measurements challenging. Dynamic Nuclear Polarization (DNP) has emerged as a method to circumvent this limitation, but most polarizing agents developed for DNP are unstable in reducing cellular environments.

Hierarchy of the Components in Spray-Dried, Protein-Excipient Particles Using DNP-Enhanced NMR Spectroscopy.

Protein-based drugs are becoming increasingly important, but there are challenges associated with their formulation (for example, formulating stable inhalable aerosols while maintaining the proper long-term stability of the protein). Determining the morphology of multicomponent, protein-based drug formulations is particularly challenging. Here, we use dynamic nuclear polarization (DNP) solid-state NMR spectroscopy to determine the hierarchy of components within spray-dried particles containing protein, trehalose, leucine, and trileucine.

H Detected Relayed Dynamic Nuclear Polarization.

Recently, it has been shown that methods based on the dynamics of H nuclear hyperpolarization in magic angle spinning (MAS) NMR experiments can be used to determine mesoscale structures in complex materials. However, these methods suffer from low sensitivity, especially since they have so far only been feasible with indirect detection of H polarization through dilute heteronuclei such as C or Si. Here we combine relayed-DNP (R-DNP) with fast MAS using 0.

In-Cell Quantification of Drugs by Magic-Angle Spinning Dynamic Nuclear Polarization NMR.

The determination of intracellular drug concentrations can provide a better understanding of the drug function and efficacy. Ideally, this should be performed nondestructively, with no modification of either the drug or the target, and with the capability to detect low amounts of the molecule of interest, in many cases in the μM to nM range (pmol to fmol per million cells). Unfortunately, it is currently challenging to have an experimental technique that provides direct quantitative measurements of intracellular drug concentrations that simultaneously satisfies these requirements.

Mg-dependent conformational equilibria in CorA and an integrated view on transport regulation.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR).

Quantification of magic angle spinning dynamic nuclear polarization NMR spectra.

Dynamic nuclear polarization (DNP) allows to dramatically enhance the sensitivity of magic angle spinning nuclear magnetic resonance (MAS NMR). DNP experiments usually rely on the detection of low-γ nuclei hyperpolarized from H with the use of cross polarization (CP), which assures more efficient signal enhancement. However, CP is usually not quantitative.

Similarities and Differences among Protein Dynamics Studied by Variable Temperature Nuclear Magnetic Resonance Relaxation.

Understanding and describing the dynamics of proteins is one of the major challenges in biology. Here, we use multifield variable-temperature NMR longitudinal relaxation (R) measurements to determine the hierarchical activation energies of motions of four different proteins: two small globular proteins (GB1 and the SH3 domain of α-spectrin), an intrinsically disordered protein (the C-terminus of the nucleoprotein of the Sendai virus, Sendai Ntail), and an outer membrane protein (OmpG). The activation energies map the motions occurring in the side chains, in the backbone, and in the hydration shells of the proteins.

Dynamic Nuclear Polarization Enhancement of 200 at 21.15 T Enabled by 65 kHz Magic Angle Spinning.

Solid-state nuclear magnetic resonance under magic angle spinning (MAS) enhanced with dynamic nuclear polarization (DNP) is a powerful approach to characterize many important classes of materials, allowing access to previously inaccessible structural and dynamic parameters. Here, we present the first DNP MAS experiments using a 0.7 mm MAS probe, which allows us to reach spinning frequencies of 65 kHz, with microwave irradiation, at 100 K.

Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR.

Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-10 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity.

The lineage-specific, intrinsically disordered N-terminal extension of monothiol glutaredoxin 1 from trypanosomes contains a regulatory region.

Glutaredoxins (Grx) are small proteins conserved throughout all the kingdoms of life that are engaged in a wide variety of biological processes and share a common thioredoxin-fold. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (FeS) metabolism. They contain a single thiol group in their active site and use low molecular mass thiols such as glutathione as ligand for binding FeS-clusters.

Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning.

Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic-angle-spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz).

Paramagnetic Properties of a Crystalline Iron-Sulfur Protein by Magic-Angle Spinning NMR Spectroscopy.

We present the first solid-state NMR study of an iron-sulfur protein. The combined use of very fast (60 kHz) magic-angle spinning and tailored radiofrequency irradiation schemes allows the detection and the assignment of most of the H and C resonances of the oxidized high-potential iron-sulfur protein I from Ectothiorhodospira halophila (EhHiPIP I), including those in residues coordinating the FeS cluster. For these residues, contact shifts as large as 100 and 400 ppm for H and C resonances, respectively, were observed, which represent the most shifted solid-state NMR signals ever measured in metalloproteins.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination.

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.

Protein residue linking in a single spectrum for magic-angle spinning NMR assignment.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning.

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems.