Residual Membrane Fluidity in Mycobacterial Cell Envelope Layers under Extreme Conditions Underlines Membrane-Centric Adaptation.

One of the routes for adaptation to extreme environments is via remodeling of cell membrane structure, composition, and biophysical properties rendering a functional membrane. Collective studies suggest some form of membrane feedback in mycobacterial species that harbor complex lipids within the outer and inner cell wall layers. Here, we study the homeostatic membrane landscape of mycobacteria in response to high hydrostatic pressure and temperature triggers using high pressure fluorescence, mass and infrared spectroscopies, NMR, SAXS, and molecular dynamics simulations. Our findings reveal that mycobacterial membrane possesses unique and lipid-specific pressure-induced signatures that attenuate progression to highly ordered phases. Both inner and outer membrane layers exhibit phase coexistence of nearly identical lipid phases keeping residual fluidity over a wide range of temperature and pressure, but with different sensitivities. Lipidomic analysis of bacteria grown under pressure revealed lipidome remodeling in terms of chain length, unsaturation, and specific long-chained characteristic mycobacterial lipids, rendering a fluid bacterial membrane. These findings could help understand how bacteria may adapt to a broad spectrum of harsh environments by modulating their lipidome to select lipids that enable the maintenance of a fluid functional cell envelope.

The Conformational Transitions and Dynamics of Burkholderia cepacia Lipase Regulated by Water-Oil Interfaces.

Structural dynamics and conformational transitions are crucial for the activities of enzymes. As one of the most widely used industrial biocatalysts, lipase could be activated by the water-oil interfaces. The interface activations were believed to be dominated by the close-to-open transitions of the lid subdomains. However, the detailed mechanism and the roles of structure transitions are still under debate. In this study, the dynamic structures and conformational transitions of Burkholderia cepacia lipase (LipA) were investigated by combining all-atom molecular dynamics simulations, enhanced sampling simulation, and spectrophotometric assay experiments. The conformational transitions between the lid-open and lid-closed states of LipA in aqueous solution are directly observed by the computational simulation methods. The interactions between the hydrophobic residues on the two lid-subdomains are the driven forces for the LipA closing. Meanwhile, the hydrophobic environment provided by the oil interfaces would separate the interactions between the lid-subdomains and promote the structure opening of LipA. Moreover, our studies demonstrate the opening of the lids structure is insufficient to initiate the interfacial activation, providing explanations for the inability of interfacial activation of many lipases with lid structures.

Mutually Exclusive Interactions of Rifabutin with Spatially Distinct Mycobacterial Cell Envelope Membrane Layers Offer Insights into Membrane-Centric Therapy of Infectious Diseases.

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane-drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

Lipid Clustering in Mycobacterial Cell Envelope Layers Governs Spatially Resolved Solvation Dynamics.

The mycobacterial cell envelope acts as a multilayered barrier to drugs. However, the role of lipid composition in the properties of different mycobacterial membranes, otherwise dictating their interactions with drugs, is poorly understood. In this study, we found that hydration states, solvation relaxation kinetics, rotational lipid mobility, and lateral lipid diffusion differed between inner and outer mycobacterial membranes. Molecular modeling showed that lipid clustering patterns governed membrane dynamics in the different layers of the cell envelope. By regulating membrane properties, lipid composition and structure modulated water abundance and interactions with lipid head groups. These findings can help deepen our understanding of the physical chemistry underlying membrane structure and function, as well as the interaction of mycobacterial membranes with drugs and host membranes.

Phosphorylation Regulation on the Homo-Dimeric Binding of Transactive Response DNA-Binding Protein.

The dimerization of transactive response DNA-binding protein of 43 kDa (TDP-43) is crucial for the RNA metabolism, and the higher-order aggregation of TDP-43 would induce several neurodegenerative diseases. The dimerization and aggregation of TDP-43 are regulated by the phosphorylation on its N-terminal domain (NTD). Understanding the regulation mechanism of TDP-43 NTD dimerization is crucial for the preventing of harmful aggregation and the associated diseases. In this study, the dimerization processes of wild-type (WT), phosphorylated S48 (pS48), and phosphomimic S48E mutation (S48E) of TDP-43 NTD are characterized by the enhanced sampling technology. Our results show that the phosphorylation not only shift the conformation population of bound and unbound state of TDP-43 NTD, but also would regulate the dimerization processes, including increase the binding free-energy barrier. The phosphomimic mutation would also shift the conformational space of TDP-43 NTD dimer to the unbound structures; however, the thermodynamic and kinetic properties of the dimerization processes between the phosphorylated and phosphomimic mutant systems are distinct, which reminds us to carefully study the phosphorylation regulation by using the phosphomimic mutations.

Screening of perhydrolases to optimize glucose oxidase-perhydrolase-in situ chemical oxidation cascade reaction system and its application in melanin decolorization.

A novel glucose oxidase (GOD)-perhydrolase-in situ chemical oxidation (ISCO) cascade reaction system was designed, optimized, and verified the operation feasibility in this research. Among the determined four perhydrolases, acyltransferase from Mycobacterium smegmatis (MsAcT) displayed the highest specific activity for perhydrolysis reaction (76.4 U/mg) and the lowest Km value to hydrogen peroxide (13.9 mmol/L). GOD-MsAcT cascade reaction system also displayed high catalytic efficiency. Under the optimal parameters (50:1 activity unit ratio of GOD to MsAcT, pH 8.0, 50 mmol/L of ß-d-glucose, and 15 mmol/L of glyceryl triacetate), the melanin decolorization rate using GOD-MsAcT-ISCO cascade reaction system reached 86.8 %. Kinetics of GOD-MsAcT-ISCO cascade reaction system for melanin decolorization fitted the kinetic model of Boltzmann sigmoid. As a substitutive skin whitening technology, GOD-MsAcT-ISCO cascade reaction system displayed an excellent application prospect.