Joseph R. Sachleben

Theoretical aspects of higher‐order truncations in solid‐state nuclear magnetic resonance

Recent experimental developments of high‐resolution NMR in solids (for example, double rotation and dynamic‐angle spinning) address the reduction of second‐order line broadening effects, particularly in systems involving quadrupolar nuclei such as 23Na, 17O, 27Al, and 14N. However, some aspects of the theoretical description of these systems have not been clearly understood; in particular, the various procedures available to truncate the interactions give incompatible results. We present a general framework, based on static perturbative methods, which provides a natural procedure to derive the correct Hamiltonian for higher‐order effects in irreducible tensor form. Applications of this method to coherent averaging techniques (sample motion or radio‐frequency irradiation) are described and compared to previous treatments based on average Hamiltonian theory.

Fast acquisition of multi-dimensional spectra in solid-state NMR enabled by ultra-fast MAS

The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored. It is shown that optimal conditions for performing solid-state (13)C NMR under 60 kHz MAS are obtained with low-power CW (1)H decoupling, as well as after a low-power (1)H,(13)C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet-van Vleck theory. As a result, optimal (13)C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short (1)H T(1)s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D (13)C-(13)C correlation could be acquired in about 24 h on less than 1 mg of SOD sample.

NMR studies of the surface structure and dynamics of semiconductor nanocrystals

H-1 NMR studies of thiophenol capping groups on cadmium sulfide nanocrystals demonstrate that the coverage of the capping molecule depends on the size of the nanocrystal. Data are presented which show that as the size of the nanocrystal increases, the coverage of thiophenol decreases. In addition, information about the overall tumbling of the nanocrystal and the motion of the capping groups relative to the surface can be obtained from linewidth studies, indicating that the rotation of the capping groups is hindered in the smaller nanocrystals (r almost-equal-to 12 angstrom) and becomes less so in larger nanocrystals (r almost-equal-to 20 angstrom). The coverage data are related to the electronic properties of this important class of compounds.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis

BNip3 is a hypoxia‐inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif‐1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3‐null tumors. Glycolysis inhibition attenuates the growth of BNip3‐null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple‐negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Use of 1H cross-relaxation nuclear magnetic resonance spectroscopy to probe the changes in bread and its components during aging

(1)H nuclear magnetic cross-relaxation spectroscopy was used to probe the molecular mobility/rigidity in bread and its components during storage. The Z-spectra lineshapes, attributed to the solid-like polymer fractions of the samples, differed for the bread, gelatinized waxy starch (GX), gelatinized wheat starch (GW), heated flour (HF), and heated gluten (HG). Upon storage, no change was observed in the Z-spectrum of the bread sample, while the Z-spectra for GX, GW, and HG increased in the width at half height of the decomposed broad component (increased rigidity). These trends in the Z-spectra detected by NMR were contradictory to the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples containing starch, including bread. These trends in the Z-spectra detected by NMR were not reflected by the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples, including bread. The differences in molecular mobility could not be therefore, due to recrystallized amylopectin and may be attributed to the role of gluten and/or redistribution of water in the amorphous regions of the samples.

The effect of spin decoupling on line shapes in solid-state nuclear magnetic resonance

Experimental and theoretical aspects of carbon‐13 line shapes in static solids are described for on‐resonance spin decoupling conditions. A relatively simple theoretical approach is provided for describing line shapes in static solids based on an operator representation of static second‐order perturbation theory and theoretical line shapes in I2S and InS systems are calculated. The line shapes are predicted to comprise a single center line and ‘‘decoupling sidebands’’ on each side of the center line which move outward and diminish in amplitude as the decoupling field is increased. The predicted behavior is confirmed by experiments on an isolated seven spin system, where the decoupling sidebands are observed directly, and some organic solids in which the decoupling sidebands are not observed directly but in which their presence can be deduced from the behavior of the center line. A comparison is made between the theoretical predictions based on a complete quantum mechanical treatment and the predictions ma...

Multi‐rank nuclear magnetic resonance studies of half‐integer quadrupolar nuclei in solids by three‐dimensional dynamic‐angle correlation spectroscopy

The present work introduces a new three‐dimensional nuclear magnetic resonance (3D NMR) experiment for the analysis of half‐integer quadrupolar nuclei in solids. The method is based on the multi‐rank expansion of the high‐field NMR Hamiltonian governing the central transition of these spins in terms of irreducible spherical tensor elements. This approach leads to a temporal spin evolution given by an isotropic term characteristic of each chemical site, as well as by second‐ and fourth‐rank anisotropies depending on the principal values and relative orientations of the shielding and quadrupolar interactions. A method for extracting the 3D spectral distribution correlating these three frequency components is presented, based on the acquisition of dynamic‐angle spinning NMR signals collected as a function of different initial and final spinning axes. Computational and instrumental details involved in the acquisition of these 3D dynamic‐angle correlation spectroscopy (DACSY) data are discussed, and applicatio...

Conformational dynamics at the inner gate of KcsA during activation.

The potassium channel KcsA offers a unique opportunity to explicitly study the dynamics of the moving parts of ion channels, yet our understanding of the extent and dynamic behavior of the physiologically relevant structural changes at the inner gate in KcsA remains incomplete. Here, we use electron paramagnetic resonance, nuclear magnetic resonance, and molecular dynamics simulations to characterize the extent of pH-dependent conformational changes of the inner gate in lipid bilayers or detergent micelles. Our results show that under physiological conditions the inner gate experiences a maximal diagonal opening of ∼24 Å with the largest degree of dynamics near the pKa of activation (pH ∼3.9). These results extend the observation that the C-terminus is necessary to limit the extent of opening and imply that the inner gate regulates the extent of conformational change at the zone of allosteric coupling and at the selectivity filter.

Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation

Significance Gamma delta T cells, a group of immune cells that exhibit features from both innate and adaptive immunity, possess significant potential in clinical applications such as treatment of microbial infections and cancer immunotherapy. To fully understand their biology and harness them in the clinic it is imperative to dissect the molecular mechanisms involved in their recognition of infected and tumor cells. In this paper we focus on Vγ9Vδ2 T cells, a major subset of human gamma delta T cells in blood and investigate the phosphoantigen-induced, MHC-independent molecular mechanisms governing their activation. Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.

Aliphatic chain length by isotropic mixing (ALCHIM): determining composition of complex lipid samples by 1H NMR spectroscopy

Quantifying the amounts and types of lipids present in mixtures is important in fields as diverse as medicine, food science, and biochemistry. Nuclear magnetic resonance (NMR) spectroscopy can quantify the total amounts of saturated and unsaturated fatty acids in mixtures, but identifying the length of saturated fatty acid or the position of unsaturation by NMR is a daunting challenge. We have developed an NMR technique, aliphatic chain length by isotropic mixing, to address this problem. Using a selective total correlation spectroscopy technique to excite and transfer magnetization from a resolved resonance, we demonstrate that the time dependence of this transfer to another resolved site depends linearly on the number of aliphatic carbons separating the two sites. This technique is applied to complex natural mixtures allowing the identification and quantification of the constituent fatty acids. The method has been applied to whole adipocytes demonstrating that it will be of great use in studies of whole tissues.