Maili Liu

Mechanism of Surfactant Micelle Formation

The mechanism of micelle formation of surfactants sodium dodecyl sulfate (SDS), n-hexyldecyltrimethylammonium bromide (CTAB) and Triton X-100 (TX-100) in heavy water solutions was studied by 1H NMR (chemical shift and line shape) and NMR self-diffusion experiments. 1H NMR and self-diffusion experiments of these three surfactants show that their chemical shifts (delta) begin to change and resonance peaks begins to broaden with the increase in concentration significantly below their critical micelle concentrations (cmcs). At the same time, self-diffusion coefficients ( D) of the surfactant molecules decrease simultaneously as their concentrations increase. These indicate that when the concentrations are near and lower than their cmcs, there are oligomers (premicelles) formed in these three surfactant systems. Carefully examining the dependence of chemical shift and self-diffusion coefficient on concentration in the region just slightly above their cmcs, one finds that the pseudophase transition model is not applicable to the variation of physical properties (chemical shift and self-diffusion coefficient) with concentration of these systems. This indicates that premicelles still exist in this concentration region along with the formation of micelles. The curved dependence of chemical shift and self-diffusion coefficient on the increase in concentration suggests that the premicelles grow as the concentration increases until a definite value when the size of the premicelle reaches that of the micelle, i.e., the system is likely dominated by the monomers and micelles. Additionally, the approximate values of premicelle coming forth concentration (pmc) and cmc were obtained by again fitting chemical shifts to reciprocals of concentrations at a different perspective, and are in good accordant with experimental results and literature values and prove the former conclusion.

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity

Aims: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase‐producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. 
Methods and Results: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty‐eight strains produced different levels of chitinase at pH 7·0, and so did 17 strains at pH 10·0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml−1 in liquid medium. SDS‐PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase‐producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2·35‐fold. 
Conclusion: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. 
Significance and Impact of the Study: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.

Metabolic changes in rat prefrontal cortex and hippocampus induced by chronic morphine treatment studied ex vivo by high resolution 1H NMR spectroscopy.

Ex vivo(1)H NMR spectroscopy was used to measure changes in the concentrations of cerebral metabolites in the prefrontal cortex (PFC) and hippocampus of rats subjected to repeated morphine treatment known to cause tolerance/dependence. The results show that repeated morphine exposure induces significant changes in the concentrations of a number of cerebral metabolites, and such changes are region specific. After 10 days of repeated morphine treatment, the concentration of gamma-aminobutyric acid (GABA) increased significantly in the PFC (20+/-11%), but decreased in the hippocampus (-31+/-12%), compared to control. In contrast, the glutamate (Glu) concentrations in both the PFC (-15+/-8%) and hippocampus (-13+/-4%) decreased significantly. Significant changes were also observed in the concentrations of hippocampal glutamine (Gln), myo-inositol, taurine, and N-acetyl aspartate. These morphine-induced changes were reversed during a subsequent 5-day withdrawal period. It is suggested that the observed concentration changes for Glu, Gln and GABA are most likely the result of a shift in the steady-state equilibrium of the Gln-Glu-GABA metabolic cycle. Changes in the metabolism of this neurotransmitter system might be part of the adaptive measures taken by the central nervous system in response to repeated morphine exposure and subsequent withdrawal.

Noncovalent Dimerization of Ubiquitin

Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5 mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.

19F NMR Spectroscopy as a Probe of Cytoplasmic Viscosity and Weak Protein Interactions in Living Cells

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of (19) F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy.

Impurity profiling in bulk pharmaceutical batches using 19F NMR spectroscopy and distinction between monomeric and dimeric impurities by NMR-based diffusion measurements.

The impurity profile of production batches of fluorine-containing drugs can be characterised efficiently using 19F NMR spectroscopy. This yields the number and proportions of impurities in the bulk drug to a level of approximately equal 0.1 mole% in a few minutes of NMR experiment time. The approach has been exemplified using a partially purified batch of the steroidal product fluticasone propionate, the impurities in which include a number of dimeric species. Further distinction between the monomer and dimer impurities has been achieved through high resolution chemical shift-resolved NMR measurement of molecular diffusion coefficients on the intact mixture using 19F NMR spectroscopy. The ability of NMR-based diffusion coefficient determination to distinguish between monomeric and dimeric substances was validated using a standard mixture of authentic materials containing both monomers and dimers.

Solution structure of all parallel G-quadruplex formed by the oncogene RET promoter sequence

RET protein functions as a receptor-type tyrosine kinase and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the promoter plays an important role in the transcriptional regulation of RET. Here, we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. The overall G-quadruplex is composed of three stacked G-tetrad and four syn guanines, which shows distinct features for all parallel-stranded folding topology. The core structure contains one G-tetrad with all syn guanines and two other with all anti-guanines. There are three double-chain reversal loops: the first and the third loops are made of 3 nt G-C-G segments, while the second one contains only 1 nt C10. These loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure and their conformations are in accord with the experimental mutations. The distinct RET promoter G-quadruplex structure suggests that it can be specifically involved in gene regulation and can be an attractive target for pathway-specific drug design.

Different Residues in Channel Turret Determining the Selectivity of ADWX-1 Inhibitor Peptide between Kv1.1 and Kv1.3 Channels

The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.

Accurately probing slow motions on millisecond timescales with a robust NMR relaxation experiment

A new pulse scheme is proposed for the accurate measurement of relaxation dispersion, which cycles the phases of CPMG pulses. Numerical simulations show that systematic errors in the measured relaxation rates mainly result from off-resonance and radio frequency inhomogeneity effects and they can be significantly suppressed with the method proposed here. The method has been demonstrated on human liver fatty acid binding protein. It allows the reliable identification of residues undergoing conformational exchange on millisecond timescales and accurate extraction of kinetics parameters. The relaxation dispersion data indicate that human liver fatty acid binding protein is highly flexible on millisecond timescales.

Creating conformational entropy by increasing interdomain mobility in ligand binding regulation: a revisit to N-terminal tandem PDZ domains of PSD-95.

The two N-terminal PDZ domains of postsynaptic density protein-95 (PDS-95 PDZ1 and PDZ2) are closely connected in tandem by a conserved peptide linker of five amino acids. The interdomain orientation between PDZ1 and PDZ2 of the ligand-free PDZ12 tandem is restrained, and this conformational arrangement facilitates the synergistic binding of PDZ12 to multimeric targets. (1) The interdomain orientation of the target-bound state of PDZ12 is not known. Here, we have solved the structure of PDZ12 in complex with its binding domain from cypin. Both chemical shift data and residual dipolar coupling measurements showed that the restrained interdomain orientation disappeared upon cypin peptide binding. NMR-based relaxation experiments revealed slow interdomain motions in the PDZ12/cypin peptide complex. Molecular dynamics simulations also showed that the PDZ12/cypin complex has larger conformational flexibility than the ligand-free PDZ12. This dramatic change of protein dynamics provides extra conformational entropy upon ligand binding, thus enhancing the ligand binding affinity of the PDZ12 tandem. Modulation of ligand binding affinity through concerted interdomain structural and dynamic rearrangements may represent a general property of multidomain scaffold proteins.