Structural Model of Bacteriophage P22 Scaffolding Protein in a Procapsid by Magic-Angle Spinning NMR.

Icosahedral dsDNA viruses such as the tailed bacteriophages and herpesviruses have a conserved pathway to virion assembly that is initiated from a scaffolding protein driven procapsid formation. The dsDNA is actively packaged into procapsids, which undergo complex maturation reactions to form infectious virions. In bacteriophage P22, scaffolding protein (SP) directs the assembly of coat proteins into procapsids that have a T=7 icosahedral arrangement, en route to the formation of the mature P22 capsid.

Integrating F Distance Restraints for Accurate Protein Structure Determination by Magic Angle Spinning NMR Spectroscopy.

Traditional protein structure determination by magic angle spinning (MAS) solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from C-, N-, and H-based dipolar-based correlation experiments. Here, we show that F fast (60 kHz) MAS NMR spectroscopy can supply additional, longer distances. Using 4F-Trp,U-C,N crystalline agglutinin (OAA), we demonstrate that judiciously designed 2D and 3D F-based dipolar correlation experiments such as (H)CF, (H)CHF, and FF can yield interatomic distances in the 8-16 Å range.

Nanodisc Reconstitution and Characterization of Amyloid-β Precursor Protein C99.

Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer's disease (AD). Since the fragmentation of the membrane-bound APP that results in the production of amyloid-β peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable and suitable membrane-mimicking lipid environment is a challenging task.

Nanodisc reconstitution and characterization of amyloid-β precursor protein C99.

Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer's disease. Since the fragmentation of the membrane-bound APP that results in the production of amyloid-beta peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable/suitable membrane-mimicking lipid environment is a challenging task.

High-efficiency low-power C-N cross polarization in MAS NMR.

Biomolecular solid-state magic angle spinning (MAS) NMR spectroscopy frequently relies on selective C-N magnetization transfers, for various kinds of correlation experiments. Introduced in 1998, spectrally induced filtering in combination with cross polarization (SPECIFIC-CP) is a selective heteronuclear magnetization transfer experiment widely used for biological applications. At MAS frequencies below 20 kHz, commonly used for C-detected MAS NMR experiments, SPECIFIC-CP transfer between amide N and C atoms (NCA) is typically performed with radiofrequency (rf) fields set higher than the MAS frequency for both C and N channels, and high-power H decoupling rf field is simultaneously applied.

Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR.

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein.

Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments.

Automation in solid state NMR.

Automation in solid state NMR (ssNMR) requires appropriate hardware, from rotor loading mechanisms over highly stable rf-transmitters and probe circuitry to automatic tuning and matching capabilities including automatic magic angle adjustment for ssNMR probes. While these hardware capabilities are highly desirable and are, to various degrees, provided by manufacturers, we focus herein on automating experiment setup using radio frequency (rf) fields, which are key parameters in solid state NMR experiments. Specifically, these include spinlock fields during cross polarization (CP), or rf-fields for homo- or heteronuclear spin recoupling or decoupling.

Overcoming challenges in Zn NMR: a new strategy of signal enhancement for MOF characterization.

Zn solid-state NMR suffers from low sensitivity, limiting its ability to probe the Zn surroundings in MOFs. We report a breakthrough in overcoming challenges in Zn NMR. Combining new cryogenic MAS probe technology and performing NMR experiments at a high magnetic field results in remarkable signal enhancement, yielding enhanced information for MOF characterization.

Exploring the Applications of Carbon-Detected NMR in Living and Dead Organisms Using a C-Optimized Comprehensive Multiphase NMR Probe.

Comprehensive multiphase-nuclear magnetic resonance (CMP-NMR) is a non-invasive approach designed to observe all phases (solutions, gels, and solids) in intact samples using a single NMR probe. Studies of dead and living organisms are important to understand processes ranging from biological growth to environmental stress. Historically, such studies have utilized H-based phase editing for the detection of soluble/swollen components and H-detected 2D NMR for metabolite assignments/screening.

Solid-state O NMR study of α-d-glucose: exploring new frontiers in isotopic labeling, sensitivity enhancement, and NMR crystallography.

We report synthesis and solid-state O NMR characterization of α-d-glucose for which all six oxygen atoms are site-specifically O-labeled. Solid-state O NMR spectra were recorded for α-d-glucose/NaCl/HO (2/1/1) cocrystals under static and magic-angle-spinning (MAS) conditions at five moderate, high, and ultrahigh magnetic fields: 14.1, 16.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site.

Racing toward Fast and Effective O Isotopic Labeling and Nuclear Magnetic Resonance Spectroscopy of N-Formyl-MLF-OH and Associated Building Blocks.

Solid-state H, C, and N nuclear magnetic resonance (NMR) spectroscopy has been an essential analytical method in studying complex molecules and biomolecules for decades. While oxygen-17 (O) NMR is an ideal and robust candidate to study hydrogen bonding within secondary and tertiary protein structures for example, it continues to elude many. We discuss an improved multiple-turnover labeling procedure to develop a fast and cost-effective method to O label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks.

Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains.

Expanding current applications and permitting the analysis of larger intact samples by means of a 7 mm CMP-NMR probe.

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors.

Comprehensive Multiphase NMR of whole organisms: A complementary tool to NMR.

Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e.