Exploring the Higher Order Structure and Conformational Transitions in Insulin Microcrystalline Biopharmaceuticals by Proton-Detected Solid-State Nuclear Magnetic Resonance at Natural Abundance.

The integrity of a higher order structure (HOS) is an essential requirement to ensure the efficacy, stability, and safety of protein therapeutics. Solution-state nuclear magnetic resonance (NMR) occupies a unique niche as one of the most promising methods to access atomic-level structural information on soluble biopharmaceutical formulations. Another major class of drugs is poorly soluble, such as microcrystalline suspensions, which poses significant challenges for the characterization of the active ingredient in its native state. Here, we have demonstrated a solid-state NMR method for HOS characterization of biopharmaceutical suspensions employing a selective excitation scheme under fast magic angle spinning (MAS). The applicability of the method is shown on commercial insulin suspensions at natural isotopic abundance. Selective excitation aided with proton detection and non-uniform sampling (NUS) provides improved sensitivity and resolution. The enhanced resolution enabled us to demonstrate the first experimental evidence of a phenol-escaping pathway in insulin, leading to conformational transitions to different hexameric states. This approach has the potential to serve as a valuable means for meticulously examining microcrystalline biopharmaceutical suspensions, which was previously not attainable in their native formulation states and can be seamlessly extended to other classes of biopharmaceuticals such as mAbs and other microcrystalline proteins.

Combined Liquid-State and Solid-State Nuclear Magnetic Resonance at Natural Abundance for Comparative Higher Order Structure Assessment in the Formulated-State of Biphasic Biopharmaceutics.

A higher-order structure (HOS) is critical to a biopharmaceutical drug as the three-dimensional structure governs its function. Even the partial perturbation in the HOS of the drug can alter the biological efficiency and efficacy. Due to current limitations in analytical technologies, it is imperative to develop a protocol to characterize the HOS of biopharmaceuticals in the native formulated state. This becomes even more challenging for the suspension formulations where solution and solid phases co-exist. Here, we have used a combinatorial approach using liquid (1D 1H) and solid-state (13C CP MAS) NMR methodology to demonstrate the HOS in the biphasic microcrystalline suspension drug in its formulated state. The data were further assessed by principal component analysis and Mahalanobis distance (DM) calculation for quantitative assessment. This approach is sufficient to provide information regarding the protein HOS and the local dynamics of the molecule when combined with orthogonal techniques such as X-ray scattering. Our method can be an elegant tool to investigate batch-to-batch variation in the process of manufacture and storage as well as a biosimilarity comparison study for biphasic/microcrystalline suspension.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Conformational Dynamics of Histone H3 Tails in Chromatin.

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.

Chirp pulse sequences for broadband π rotation.

In this paper, we present various chirp pulse sequences for implementing a broadband π rotation, which can serve as an ideal refocusing pulse element in a spin echo pulse sequence. These sequences are composed of three pulse elements each of which do adiabatic passage at rate a or 2a in either backward or forward direction. Various possible combinations are considered and different variants are presented. They all implement a broadband π rotation. We present the theory of such composite chirp sequences along with simulations and experiments.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-µs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

Probing the gel to liquid-crystalline phase transition and relevant conformation changes in liposomes by (13)C magic-angle spinning NMR spectroscopy.

A straightforward way to visualize gel to liquid-crystalline phase transition in phospholipid membranes is presented by using ¹³C magic-angle spinning NMR. The changes in the 13C isotropic chemical shifts with increasing temperature are shown to be a sensitive probe of the main thermotropic phase transition related to lipid hydrocarbon chain dynamics and relevant conformational changes. The average value of the energy difference between trans and gauche states in the central C4­11 fragment of the DMPC acyl chain was estimated to be 4.02 ± 0.2 kJ mol⁻¹ in the liquid crystalline phase. The reported spectral features will be useful in 13C solid state NMR studies for direct monitoring of the effective lipid chain melting allowing rapid uniaxial rotation of membrane proteins in the biologically relevant liquid-crystalline phase.

Determination of sample temperature in unstable static fields by combining solid-state (79)Br and (13)C NMR.

Monitoring the isotropic chemical shifts to calibrate the sample temperature presupposes a perfect stability of the static magnetic field. It can be difficult to satisfy this requirement in solid-state NMR measurements. This paper describes a simple way to recover the accurate temperature dependence of the (79)Br resonance after subtracting changes of resonance frequency due to variations of the static field, monitored by the (13)C resonance.

Abnormal lipid metabolism in collagen-induced arthritis rat model: in vitro, high resolution NMR spectroscopy based analysis.

Collagen-induced arthritis (CIA) was induced in female Wistar rats by intradermal injection of porcine immunization grade native collagen type II (Chondrex). Development and progression of CIA was monitored by studying histopathological, radiographical and biochemical features of arthritic manifestations in the knee joints, hind limb and blood plasma. In addition, oxidative stress status of arthritic animals was determined by measuring lipid peroxidation and the antioxidant enzymes: catalase, superoxide dismutase and glutathione peroxidase. High resolution proton NMR spectroscopy was employed for the analysis of lipid components in the lipid extracts of the joint tissue and plasma of collagen-induced arthritic and control rats. Triglyceride levels showed significant decreases in plasma (1.7 times) but were unchanged in the joint tissue of CIA rats as compared to control. One-dimensional proton NMR spectra showed a 6.2 times reduction in the quantity of choline-containing phospholipids in the plasma of CIA as compared to control rats. There was a 1.6 times elevation of choline-containing phospholipids in the joint tissue of CIA rats as compared to controls. Induction of arthritis showed a 4.0 times reduction in the level of total cholesterol in the plasma and 1.6 times elevation in the joint tissue of CIA rats as compared to controls. The ratio of saturated fatty acids to unsaturated fatty acids was 1.5 times significantly higher in joint tissue and 2.1 times significantly higher in plasma of CIA rats as compared to controls. The results demonstrated significantly altered lipid patterns in the joint tissue and plasma of collagen-induced arthritic rats as detected by one- and two-dimensional NMR spectroscopy compared with controls.

Quantitative one- and two-dimensional 13C spectra of microcrystalline proteins with enhanced intensity.

We recorded quantitative, uniformly enhanced one- and two-dimensional (13)C spectra of labelled microcrystalline proteins. The approach takes advantage of efficient equilibration of magnetization by low-power proton irradiation using Phase Alternated Recoupling Irradiation Schemes and benefits simultaneously from uniform sensitivity enhancement due to efficient spin exchange that can overcome T1((13)C) constraints and the presence of heteronuclear Overhauser effects.

Mechanistic insights into water-protein interactions of filamentous bacteriophage.

Water plays a major structural and functional role around proteins. In an attempt to explore this mechanistic structural aspect of proteins, we present site-specific interaction of hydration water with the major coat protein subunit of filamentous virus Pf1 by magic angle spinning (MAS) solid-state NMR. The interaction of surrounding water with 36 MDa Pf1 virion is investigated in uniformly (13)C, (15)N isotopically labeled; polyethylene glycol precipitated fully hydrated samples by solid-state nuclear magnetic resonance spectroscopy. Dipolar edited two-dimensional (2D) (1)H-(15)N heteronuclear correlation (HETCOR) experiments lead to unambiguous assignments of cross-peaks originating exclusively from (1)H resonances of water molecules correlating to the protein amide nitrogen. An enhanced resolved (1)H chemical shift dimension in these experiments also precludes the need of perdeuteration. We report seven residues spanning the 40-residue continuous α-helical conformation assembly of Pf1 interacting with surrounding water. It shows a highly hydrated inner core inside this viral filamentous assembly. The results obtained also suggest the first evidence of a water-mediated interface cluster formed at the site of Arg44 with the single-stranded DNA genome of the filamentous phage supramolecular assembly.

Metabonomic study of host-phage interaction by nuclear magnetic resonance- and statistical total correlation spectroscopy-based analysis.

We present a method for the qualitative and quantitative study of transient metabolic flux of phage infection at the molecular level. The method is based on statistical total correlation spectroscopy (STOCSY) and partial least squares discriminant analysis (PLS-DA) applied to nuclear magnetic resonance (NMR) metabonomic data sets. An algorithm for this type of study is developed and demonstrated. The method has been implemented on (1)H NMR data sets of growth media in planktonic cultures of Pseudomonas aeruginosa infected with bacteriophage pf1. Transient metabolic flux of various important metabolites, identified by STOCSY and PLS-DA analysis applied to the NMR data set, are estimated at various stages of growth. The opportunistic and nosocomial pathogen P. aeruginosa is one of the best-studied model organism for bacterial biofilms. Complete information regarding metabolic connectivity of this system is not possible by conventional spectroscopic approach. Our study presents temporal comparative (1)H NMR metabonomic analyses of filamentous phage pf1 infection in planktonic cultures of P. aeruginosa K strain (PAK). We exemplify here the potential of STOCSY and PLS-DA tools to gain mechanistic insight into subtle changes and to determine the transient flux associated with metabolites following metabolic perturbations resulting from phage infection. Our study has given new avenues in correlating existing postgenomic data with current metabonomic results in P. aeruginosa biofilms research.