Project description:The translational dynamics of xenon gas dissolved in room-temperature ionic liquids (RTILs) is revealed by 129Xe NMR and molecular dynamics (MD) simulations. The dynamic behavior of xenon gas loaded in 1-alkyl-3-methylimidazolium chloride, [CnC1im]Cl (n = 6, 8, 10), and hexafluorophosphate, [CnC1im][PF6] (n = 4, 6, 8, 10) has been determined by measuring the 129Xe diffusion coefficients and NMR relaxation times. The analysis of the experimental NMR data demonstrates that, in these representative classes of ionic liquids, xenon motion is influenced by the length of the cation alkyl chain and anion type. 129Xe spin-lattice relaxation times are well described with a monoexponential function, indicating that xenon gas in ILs effectively experiences a single average environment. These experimental results can be rationalized based on the analysis of classical MD trajectories. The mechanism described here can be particularly useful in understanding the separation and adsorption properties of RTILs.
Project description:A lipopolysaccharide (LPS) molecule is a key component of the bacterial outer membrane used to protect the bacterium and to interact with the environment. To gain insight into its function, the study of the LPS conformation and dynamics at the molecular and cellular levels is necessary, but these highly diverse and dynamic membrane-LPS systems are difficult to study. In this work, by using NMR spectroscopy and molecular dynamics (MD) simulations, we determined the conformational preferences of an E. coli O176 O-antigen polysaccharide at the atomic level. Moreover, we analyzed the use of non-uniform sampling (NUS) for the acquisition of high dynamic range spectra, like 1H,1H-NOESY NMR experiments. A comparison of the effective transglycosidic distances derived from conventional uniformly sampled and NUS 1H,1H-NOESY data showed high similarity under equal measuring time conditions. Furthermore, the experimentally derived internuclear distances of the O-antigen polysaccharide with ten repeating units (RUs) showed very good agreement to those calculated from the MD simulations of the same O-antigen polysaccharide in solution. Analysis of the LPS bilayer simulations with five and with ten RUs revealed that, although similar with respect to populated states in solution, the O-antigen in LPS bilayers had more extended chains as a result of spatial limitations due to close packing. Additional MD simulations of O-antigen polysaccharides from E. coli O6 (branched repeating unit) and O91 (negatively charged linear repeating unit) in solution and LPS bilayers were performed and compared to those of O176 (linear polymer). For all three O-antigens, the ensemble of structures present for the polysaccharides in solution were consistent with the results from their 1H,1H-NOESY experiments. In addition, the similarities between the O-antigen on its own and as a constituent of the full LPS in bilayer environment makes it possible to realistically describe the LPS conformation and dynamics from the MD simulations.
Project description:The replica exchange molecular dynamics (REMD) simulation is demonstrated to readily predict the conformations of the macrocyclic drug lorlatinib, as validated by solution NMR studies. In aqueous solution, lorlatinib adopts a conformer identical to its target bound structure. This conformer is stabilized by an extensive hydrogen bond network to the solvents. In chloroform, lorlatinib populates two conformers with the second one being less polar, which may contribute to lorlatinib's ability to cross cell membranes.
Project description:Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin β cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the α/β transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions.
Project description:We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape.
Project description:The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe3049 and Trp3052) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.
Project description:Nuclear magnetic resonance (NMR) relaxation data and molecular dynamics (MD) simulations are combined to characterize the dynamics of the fungal prion HET-s(218-289) in its amyloid form. NMR data is analyzed with the dynamics detector method, which yields timescale-specific information. An analogous analysis is performed on MD trajectories. Because specific MD predictions can be verified as agreeing with the NMR data, MD was used for further interpretation of NMR results: for the different timescales, cross-correlation coefficients were derived to quantify the correlation of the motion between different residues. Short timescales are the result of very local motions, while longer timescales are found for longer-range correlated motion. Similar trends on ns- and μs-timescales suggest that μs motion in fibrils is the result of motion correlated over many fibril layers.
Project description:Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.
Project description:Interactions of transmembrane receptors with their extracellular ligands are essential for cellular communication and signaling and are therefore a major focus in drug discovery programs. The transition from in vitro to live cell interaction studies, however, is typically a bottleneck in many drug discovery projects due to the challenge of obtaining atomic-resolution information under near-physiological conditions. Although NMR spectroscopy is ideally suited to overcome this limitation, several experimental impairments are still present. Herein, we propose the use of methylcellulose hydrogels to study extracellular proteins and their interactions with plasma membrane receptors. This approach reduces cell sedimentation, prevents the internalization of membrane receptors, and increases cell survival, while retaining the free tumbling of extracellular proteins.