Biochim Biophys Acta Biomembr
October 2024
Cell membranes are responsible for a range of biological processes that require interactions between lipids and proteins. While the effects of lipids on proteins are becoming better understood, our knowledge of how protein conformational changes influence membrane dynamics remains rudimentary. Here, we performed experiments and computer simulations to study the dynamic response of a lipid membrane to changes in the conformational state of pH-low insertion peptide (pHLIP), which transitions from a surface-associated (SA) state at neutral or basic pH to a transmembrane (TM) α-helix under acidic conditions.
View Article and Find Full Text PDFThe delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied.
View Article and Find Full Text PDFTransient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species.
View Article and Find Full Text PDFPredicting the range of substrates accepted by an enzyme from its amino acid sequence is challenging. Although sequence- and structure-based annotation approaches are often accurate for predicting broad categories of substrate specificity, they generally cannot predict which specific molecules will be accepted as substrates for a given enzyme, particularly within a class of closely related molecules. Combining targeted experimental activity data with structural modeling, ligand docking, and physicochemical properties of proteins and ligands with various machine learning models provides complementary information that can lead to accurate predictions of substrate scope for related enzymes.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
September 2020
Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.
View Article and Find Full Text PDFDeveloping cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of .
View Article and Find Full Text PDFElucidating the interaction networks associated with secondary metabolite production in microorganisms is an ongoing challenge made all the more daunting by the rate at which DNA sequencing technology reveals new genes and potential pathways. Developing the culturing methods, expression conditions, and genetic systems needed for validating pathways in newly discovered microorganisms is often not possible. Therefore, new tools and techniques are needed for defining complex metabolic pathways.
View Article and Find Full Text PDFSpatial and temporal profiling of metabolites within and between living systems is vital to understanding how chemical signaling shapes the composition and function of these complex systems. Measurement of metabolites is challenging because they are often not amenable to extrinsic tags, are diverse in nature, and are present with a broad range of concentrations. Moreover, direct imaging by chemically informative tools can significantly compromise viability of the system of interest or lack adequate resolution.
View Article and Find Full Text PDFFreely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function.
View Article and Find Full Text PDFMetabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction.
View Article and Find Full Text PDFLiving systems possess a rich biochemistry that can be harnessed through metabolic engineering to produce valuable therapeutics, fuels and fine chemicals. In spite of the tools created for this purpose, many organisms tend to be recalcitrant to modification or difficult to optimize. Crude cellular extracts, made by lysis of cells, possess much of the same biochemical capability, but in an easier to manipulate context.
View Article and Find Full Text PDFIndole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that Pantoea sp. YR343, a microbe isolated from Populus deltoides, synthesizes IAA using the indole-3-pyruvate (IPA) pathway.
View Article and Find Full Text PDFCell-free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins, while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell-free extracts used to carry out synthesis are generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. We explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions.
View Article and Find Full Text PDFLipid extracts are an excellent choice of model biomembrane; however at present, there are no commercially available lipid extracts or computational models that mimic microbial membranes containing the branched-chain fatty acids found in many pathogenic and industrially relevant bacteria. We advance the extract of Bacillus subtilis as a standard model for these diverse systems, providing a detailed experimental description and equilibrated atomistic bilayer model included as Supporting Information to this Letter and at ( http://cmb.ornl.
View Article and Find Full Text PDFBacteriophage T4 lysozyme (T4L) has been used as a paradigm for seminal biophysical studies on protein structure, dynamics, and stability. Approximately 700 mutants of this protein and their respective complexes have been characterized by X-ray crystallography; however, despite the high resolution diffraction limits attained in several studies, no hydrogen atoms were reported being visualized in the electron density maps. To address this, a 2.
View Article and Find Full Text PDFExamining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity.
View Article and Find Full Text PDFDiverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections.
View Article and Find Full Text PDFCholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature.
View Article and Find Full Text PDFPrimary alcohols can be deoxygenated cleanly and in yields of 60-95% by reduction of derived diphenyl phosphate esters with lithium triethylborohydride in tetrahydrofuran at room temperature. Selective deoxygenation of a primary alcohol in the presence of a secondary alcohol was demonstrated. The two-step process can be performed in one pot, making it simple and convenient.
View Article and Find Full Text PDFCell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress.
View Article and Find Full Text PDFEffective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species.
View Article and Find Full Text PDFThe lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains.
View Article and Find Full Text PDFAll three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABACR (GABA(A)-ρ), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABACR versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABACR(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABACR antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABAAR antagonist, SR95531; GABABR antagonist, CGP, and agonist, baclofen.
View Article and Find Full Text PDFLignin is a complex, branched polymer that reinforces plant tissue. Understanding the factors that govern lignin structure is of central importance to the development of technologies for converting lignocellulosic biomass into fuels because lignin imparts resistance to chemical, enzymatic, and mechanical deconstruction. Lignin is formed by enzymatic oxidation of phenolic monomers (monolignols) of three main types, guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) subunits.
View Article and Find Full Text PDFNanometer-scale domains in cholesterol-rich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain.
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