The substitution reaction of Cl− ion in the complex [IrCl6]2− with the acetone molecule was investigated in detail by CW X-band EPR and UV–Vis spectroscopy methods. An original software package for deconvolution of a series of EPR or optical spectra has been developed. Based on the all-data analysis, the most probable mechanism of the process under study is proposed, including four reactions (two reversible ones). One of the stages is the redox reactions between iridium(IV) and iridium(III).
Sickle-Cell Disease (SCD) is one of the most common autosomal recessive genetic blood disorders that manifest in abnormal behavior of the red blood cells (RBCs). The mutated Hb causes sickling of RBCs under deoxygenated conditions, reducing their flowing ability, pliability, and resulting in hemolysis. The pathophysiology observed in the Indian cohort varies regionally, with some Indian tribal populations depicting milder symptoms despite SCD being relatively prevalent among them. To understand the pathogenesis of SCD with respect to nongenetic parameters, we initiated a comparative untargeted metabolomics study of the eastern Indian cohort of SCD patients using 1H NMR spectroscopy. In this exploratory study, we focused only on a small cohort of 26 SCD patients from the eastern part of India with relatively high prevalence of SCD. Our NMR-based metabolomics, in combination with statistical analyses, yielded 11 of the 29 identified metabolites that showed a statistically significant difference in concentrations between healthy controls and SCD patients. The dysregulated metabolites include molecules involved in glycolysis, hypoxic, and acute-stress conditions.
Deamidation, a modification of glutamine residues in host proteins, plays a key role in bacterial pathogenesis, where bacterial effectors manipulate host signaling pathways by modifying proteins like ubiquitin (Ub) and Ub-like protein NEDD8. The study uses traditional and real-time NMR-based techniques to investigate the enzymatic activity of two bacterial deamidases, cycle inhibitory factors, CIFEC from Escherichia coli and CIFBP from Burkholderia pseudomallei. We employed BEST-HSQC NMR spectroscopy to monitor real-time deamidation of ubiquitin, providing a robust and efficient method for quantifying enzyme activity. Our findings highlight significant differences in catalytic efficiency between CIFEC and CIFBP, despite their structural similarities. NMR-based rate measurements show CIFBP has higher catalytic efficiency than CIFEC, consistent with the previous reports, while kinetic analysis of CIFEC indicates relatively weak substrate binding and suboptimal efficiency, suggesting a potential regulatory role during infections. While the overall globular fold of the ubiquitin remains unchanged upon deamidation, we observed changes in the local chemical environment, suggesting potential localized structural changes. This study extends the use of NMR spectroscopy to investigate irreversible posttranslational modifications (PTMs) like deamidation, offering a valuable tool for understanding the molecular mechanisms behind bacterial manipulation of host cellular processes.
Small heat shock proteins (sHSPs) are essential molecular chaperones that play a crucial role in maintaining protein homeostasis and protecting cells from stress-induced damage. HSPB8 (Small heat shock protein B8), in particular, plays a crucial role in protein folding and degradation pathways and has been associated with protein aggregation disorders. However, its structural and dynamic behavior under different environmental stress conditions remains poorly defined. In particular, the effect of pH, temperature, and concentration on its oligomeric state and structural integrity needs further investigation. In this study, we performed the biophysical characterization of full-length HSPB8 and its α-crystallin domain (ACD) using solution-state nuclear magnetic resonance (NMR) spectroscopy under different environmental perturbations. The effect on the monomer–dimer equilibrium of the ACD was characterized by monitoring changes in chemical shifts and linewidths in response to the perturbations, including protein concentration, pH, and temperature. It was observed that at low pH, reduced protein concentrations, and elevated temperatures, the ACD favored sharper resonances, reflecting a monomeric state. The relative contribution of disulfide bond and noncovalent interactions in stabilizing the dimer form of ACD was also established. These results provide NMR-based molecular insights into the dynamic monomer–dimer equilibrium, crucial for HSPB8 function in protein folding.
Two empirical equations have been devised to transform absolute shieldings (GIAO) into chemical shifts. Both correlations are excellent, but that based on M06-2X/aug-cc-pVDZ calculations needs a correction term for chlorine-substituted boron compounds with B–Cl bonds. These equations have been tested against a collection of closoboranes, neutrals, and dianionic.
Simulations are essential in NMR spectroscopy for understanding and quantitatively predicting the outcomes of experiments. Dynamic NMR experiments are influenced by relaxation and chemical exchange phenomena, which complicate their interpretation. The quantum mechanical theory behind NMR simulations is well established and enables simulations under various conditions or states of the system. In particular, the representation of the spin system in terms of the density matrix, energy and pulse operators, and relaxation and chemical exchange in Liouville space is straightforward and allows for the routine simulation of dynamic NMR experiments. In fact, several advanced open-source software packages are currently available—such as Spinach, GAMMA (pyGAMMA), Simpson, ChemEx, and NMR TITAN—which users can choose from based on their simulation needs. Many of these software packages are optimized for computational speed and make use of sophisticated bases, such as the irreducible spherical tensor basis. Here, we present simulations using the direct method (spin product basis), as implemented in GAMMA, which we believe will help users better understand the underlying theory and thereby explore the other available software resources more effectively. We chose two experiments—CPMG-RD and CEST—as a case study to understand the conformational changes in proteins or macromolecules occurring in the fast and slow exchange regimes, which are frequently investigated by structural biologists.
A new protocol based on the 3D-hNCAnH experiment for rapid, unambiguous backbone resonance assignment and estimation of structural information by intensity quantification of the peaks is described here. Along F1−F3 plane at F2(13Cαi) of the 3D-hNCAnH spectrum, each i to i + 1 sequential connectivity (i.e., HiNi → Hi + 1Ni + 1) is confirmed by two inter-residue sequential correlation peaks: HiNi + 1 and Hi + 1Ni. This allows unambiguous and direct identification of sequential correlations in HSQC peaks, without the need for extensive searching in different planes of 3D spectra. Further, a protocol utilizing the ratio of the intensities of the diagonal and cross peaks along F3(1H) dimension centered at self F2(13Cαi) and sequential F2(13Cαi-1) chemical shifts taken from F2–F3 plane at F1(15Ni) of the 3D-hNCAnH spectrum for estimating one and two bond N–Cα J-coupling constants, respectively, is described. The reliability of this approach is demonstrated using doubly labelled ubiquitin protein, wherein the coupling constants that are measured by the method described here are compared with previously measured values (BMRB Nos.: 15907 and 16582). The application of the approach to other proteins is demonstrated using doubly labelled human SUMO and Ca2+ bound M-crystallin proteins.
One-dimensional quantitative NMR (qNMR) has been predominantly applied to accurately quantify pharmaceutical compounds. However, its application to complex molecules is limited due to several challenges, such as signal response overlap and spectral complexity in 1H qNMR, prolonged acquisition times for 13C, and the potential absence of relevant nuclei for 19F or 31P NMR. In 1H qNMR, complex overlapping NMR peaks in a spectrum hinder quantification by restricting the selection of integration ranges. Specifically, selecting the correct signal response for integration is a major limitation. For example, while developing 1H qNMR methods, signal responses of the target analyte and impurities could occur in the same chemical shift region, leading to measurement errors. Nowadays, two-dimensional (2D) qNMR is emerging as a viable quantitative technique to overcome such limitations. In this work, the application of the 2D NMR method (1H-13C heteronuclear single-quantum coherence spectroscopy [HSQC]) was established to perform quantitative analysis for candesartan cilexetil, thus allowing a more precise quantitation method for a spectrum with complex signal patterns. Quantifying candesartan cilexetil using a 2D 1H-13C HSQC experiment gives a good correlation between the measured and the actual sample weight. The study confirmed that the purity and sample weight can be measured accurately. The present study concludes that advanced NMR approaches can be utilized for the quantification of complex molecules. These advanced 2D qNMR approaches can be extended to other complex molecules and adopted using low-field benchtop instruments.
Saturation transfer difference (STD) NMR is a robust, versatile technique for detecting small molecules binding to large receptors. In addition to identifying binding molecules in the presence of nonbinding molecules, the STD-NMR technique can be used to determine epitope maps and binding constants. In recent years, this technique has been applied to small molecules interacting with nanoparticles. In this tutorial, we introduce the technique of STD-NMR and how it can be used to gain information about small molecules interacting with nanoparticle surfaces. After describing the principle of the STD-NMR technique, we will explain how to best prepare the sample, set up the experiment, and analyze the resulting data when nanoparticles are involved. We will also present extensions to the STD-NMR technique, alternative approaches for when STD-NMR is not ideal, and future directions for the field.
We reveal contrasting behaviors in molecular motion between the two materials, including the identification of resonance-enhanced dynamic features in elastomers. We present a depth-resolved analysis of molecular dynamics in semicrystalline polytetrafluoroethylene (PTFE) and fully amorphous fluorinated elastomer (SIFEL) films using static-gradient solid-state 19F NMR imaging. By measuring spin–lattice relaxation rates ( ) at multiple frequencies and evaluating the corresponding spectral density functions, we reveal distinct dynamic behaviors between the two materials. PTFE exhibits pronounced depth dependence in , indicating enhanced molecular motion near the surface due to a structurally disordered amorphous layer. In contrast, the fluorinated elastomer shows spatially uniform values, reflecting its homogeneous molecular mobility. Notably, the elastomer's spectral density function contains resonance-like peaks at finite frequencies, suggesting the presence of intrinsic vibrational modes superimposed on stochastic motion. This hybrid dynamic signature, captured through nuclear magnetic resonance (NMR) relaxation, offers a unique fingerprint of the elastomer's viscoelastic behavior. Our results demonstrate that static-gradient NMR imaging can probe subtle spatial variations in polymer dynamics noninvasively and with high sensitivity, enabling direct comparison between crystalline and amorphous systems. The findings provide new insights into nanoscale surface dynamics and contribute to the development of advanced materials with tailored thermomechanical properties.
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：