Quantitative analysis of multisite interactions between a protein and its binding partner at atomic resolution is complicated because locating the binding sites is difficult and differentiating the flexibility of each binding site is even more elusive. Introduction of a paramagnetic metal center close to the binding pocket greatly attenuates the signals in the NMR spectrum upon binding. Herein, the multisite binding of hen egg white lysozyme (HEWL) with lanthanide complexes [Ln(DPA)3]3− (DPA=dipicolinic acid) was analyzed with sensitive paramagnetic NMR spectroscopy. Paramagnetic relaxation enhancement (PRE) revealed that HEWL interacts with [Ln(DPA)3]3− at four major binding sites in aqueous solution, which is in contrast to a previous X-ray structural analysis. The varied binding affinities for the ligands and different flexibilities at each binding site were in good agreement with atomistic molecular dynamics (MD) simulations. The present work demonstrates that a combination of paramagnetic NMR spectroscopy and MD simulations is a powerful tool to delineate the multisite interactions of a protein with its binding partner at atomic resolution, in terms of both affinity and flexibility.
Abstract The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition‐like thiol–ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent‐exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl‐substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L ‐cysteine was evaluated by UV absorption and high‐resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent‐exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.
The quantitative relationship between the surface chemistry of carbon materials and the compatibility with polymers is a fundamental and vital physical chemistry problem in the field of polymer nanocomposites. Traditional experimental methods are difficult to solve this problem, so no theory has been formed to guide the functionalization of carbon materials. In this work, the quantitative relationship between functional groups and Hildebrand (δT) and transformed Hansen (δvdW and δele) solubility parameters of fullerene (C60) was determined by molecular dynamics simulation. Besides, which solubility parameter can more accurately predict the compatibility between C60 and three typical polymers with different polarity as a function of grafting ratio is investigated. Very interestingly, no matter which group is grafted, δT and δvdW of C60 show a slight increase first and then a decrease with the grafting ratio, whereas δele first increases abruptly and then decreases slightly. The introduction of polar groups (-OH, -COOH, and -NH2) is conducive to improving the compatibility between C60 and polymers, whereas the introduction of the nonpolar group (-CH3) is not. In terms of predicting compatibility, the Hildebrand solubility parameter is better than the Hansen solubility parameter due to the nonpolar nature of the polymers, even for nitrile butadiene rubber. Finally, the optimum grafting ratios corresponding to the maximum binding energies of C60/polymers mixtures were obtained. This study provides a new understanding of the functionalization of C60 at the molecular level and promotes the development of the theory of the thermodynamics of mixing.
An eco-friendly and practical method for the ultrasonic multicomponent synthesis of diverse (Z)-β-iodo vinylthiocyanates from cheap and commercially available alkynes, molecular iodine, and KSCN through an intermolecular H-bonding assistance strategy was developed.
The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.
Enantiomeric analysis is of great significance in chemistry, chemical biology and pharmaceutical research. We herein propose a chiral 19F NMR tag containing an amino reactive NHS group to discriminate the enantiomeric amino acids under physiological conditions by NMR spectroscopy. The chiral 19F NMR tag readily forms stable amide products with the amino acids in aqueous solution. The stereospecific chemistry of enantiomeric amino acids is discriminated by a stereogenic carbon bonded with a 19F atom and is therefore decoded by the 19F reporter and manifested in the distinct 19F chemical shift. The chemical shift difference (ΔΔδ) of the chiral 19F NMR tag derived enantiomeric amino acids variants has a broad chemical shift range between -1.13 to 1.68 ppm, indicating the high sensitivity of the chiral 19F NMR tag to the stereospecific environment surrounding the individual amino acids. The distinguishable chemical shift produced by the chiral 19F NMR tag permits simultaneous discrimination and quantification of the enantiomeric amino acids in a mixture. The high fidelity of the chiral 19F NMR tag affords high-accuracy determination of the enantiomeric composition of amino acids by simple 1D NMR under physiological conditions.
A larger number of studies successfully prepared various polymer materials with excellent self-healing properties, but the study on the underlying self-healing mechanism remains comparably backward and still unclear. In this study, we prepared a self-healing polyurethane-urea (PUU) elastomer based on noncovalent bonds. Then, a coarse-grained model of PUU was successfully constructed using the iteration Boltzmann inversion (IBI) method. Microphase separation and mechanical properties of PUU were reproduced using this model by coarse-grained molecular dynamics (MD) simulation. The three-stage healing mechanism comprised the following: (1) movement of the material to close the gap, (2) interdiffusion of the polymer, and (3) bond exchange. The mechanism was revealed by determining the effects of hard segment content on the microstructure (chain entanglement, interactions of soft and hard segments, chain motility) and healing capacity over healing time. In the initial stage of healing, the polymer chains were disentangled, and the degree of entanglement of the healed samples decreased. A novel experimental strategy confirmed the transition of hydrogen bonds from disorder to order during the healing process. The motility of the cut polymer chains (low molecular weight), especially the cut soft segment, and the disordered hydrogen bonds played a key role in the healing capacity. The increased content of the ordered hydrogen bonds led to the formation of a hard segment network, which was not conducive to healing. Finally, the promoting mechanism of external factors, such as heating and trace amount of solvent, on the healing of PUU was explained. Our work systematically and profoundly reveals the self-healing behavior and mechanism of microphase-separated PUU at the molecular level.
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