Berntsencrowley9047

Proteins present a significant challenge for nanopore-based sequence analysis. This is partly due to their stable tertiary structures that must be unfolded for linear translocation, and the absence of regular charge density. To address these challenges, here we describe how ClpXP, an ATP-dependent protein unfoldase, can be harnessed to unfold and processively translocate multi-domain protein substrates through an alpha-hemolysin nanopore sensor. This process results in ionic current patterns that are diagnostic of protein sequence and structure at the single-molecule level.Nanopore technology enables the detection and analysis of single protein molecules. The technique measures the ionic current passing through a single pore inserted in an electrically insulating membrane. The translocation of the protein molecule through the pore causes a modulation of the ionic current. Analysis of the ionic current reveals the biophysics of co-translocational unfolding and may be used to infer the amino acid sequence and posttranslational modifications of the molecule.Many enzymatic activity assays are based on either (1) identifying and quantifying the enzyme with methods such as western blot or enzyme-linked substrate assay (ELISA) or (2) quantifying the enzymatic reaction by monitoring the changing levels of either product or substrate. We have generated an outer membrane protein G (OmpG)-based nanopore approach to distinguish enzyme identity as well as analyze the enzyme's catalytic activity. Here, we engineered an OmpG nanopore with a peptide cut site inserted into one of its loops to detect proteolytic behavior. In addition, we generated an OmpG nanopore with a single-stranded DNA attached to a loop for analyzing nucleolytic cleavage. This OmpG nanopore approach may be highly useful in analyzing specific enzymes in complex biological samples, or in directly determining kinetics of enzyme-substrate complex association and dissociation.Nanopore enzymology is a powerful single-molecule technique for the label-free study of enzymes using engineered protein nanopore sensors. The technique has been applied to protein kinases, where it has enabled the full repertoire of kinase function to be observed, including kinetics of substrate binding and dissociation, product binding and dissociation, nucleotide binding, and reversible phosphorylation. Further, minor modifications enable the screening of type I kinase inhibitors and the determination of inhibition constants in a facile and label-free manner. Here, we describe the design and production of suitably engineered protein nanopores and their use for the determination of key mechanistic parameters of kinases. We also provide procedures for the determination of inhibition constants of protein kinase inhibitors.Nanopore sensing is a powerful lab-on-a-chip technique that allows for the analysis of biomarkers present in small sample sizes. In general, nanopore clogging and low detection accuracy arise when the sample becomes more and more complex such as in blood or lysate. To address this, we developed an OmpG nanopore that distinguishes among not only different proteins in a mixture but also protein homologs. AS101 purchase Here, we describe this OmpG-based nanopore system that specifically analyzes targets biomarkers in complex mixtures.Antibacterial resistance (AR) is causing more and more bacterial infections that cannot be cured by using the antibacterial drugs that are currently available. It is predicted that 10 million people will die every year by 2050 from infections caused by antibacterial resistant strains, surpassing the predicted numbers of deaths caused by cancer. AR is therefore a global challenge and novel antibacterial strategies are in high demand. To this end, the work on exploring the pore properties of a bacterial sugar transporter, WzaK30, has led to the discovery of the first inhibitor against bacterial capsular polysaccharides export.Recently, single-molecule recapitulation of capsular polysaccharide (CPS) export and pore formation properties of Wza barrel peptides have also revealed the possibility of a next-generation of Wza strategies. These strategies are based upon the first examination and understanding of the pore properties of wild-type (WT) and mutant WzaK30 in single-molecule electrical channel recording. The initially reported experimental procedures have been further developed to enable efficient studies of other Wza homologs that are more common in bacterial pathogens causing significant bacterial infections. Therefore, this chapter presents the most recent protocols and logistics behind the research on Wza channel activity, antibacterials, and strategies. The disciplines covered here include computation, molecular biology, biochemistry, electrophysiology, microbiology, and biophysics.Single-channel planar lipid bilayer (PLB) recording of bacterial porins has revealed molecular details of transport across the outer membrane of Gram-negative bacteria, including antibiotic permeation and protein translocation. To explore directional transport processes across cellular membranes, the orientation of porins or other pore-forming proteins must be established in a lipid bilayer prior to experimentation. Here, we describe a direct method for determining the orientation of porins in a PLB-with a focus on E. coli OmpF-by using targeted covalent modification of cysteine mutants. Each of the two possible orientations can be correlated with the porin conductance asymmetry, such that thereafter an I-V curve taken at the start of an experiment will suffice to establish orientation.Versatile lipid membrane-inserting nanopores have been made by functionalizing DNA nanostructures with hydrophobic tags. Here, we outline design and considerations to obtain DNA nanopores with the desired dimensions and conductance properties. We further provide guidance on their reconstitution into lipid membranes.Membrane protein pores have demonstrated applications in nanopore technology. Previous studies have mostly focused on β-barrel protein pores, whereas α-helix-based transmembrane protein pores are rarely explored in nanopore applications. Here, we developed a synthetic transmembrane peptide pore built entirely from short synthetic α-helical peptides. We examined the formation of a stable uniform ion-selective pore in single-channel electrical recordings. Furthermore, we show that cyclodextrins (CDs) block the peptide pores and determine the kinetics of CD binding and translocation. We suggest that such designed synthetic transmembrane pores will be useful for several applications in biotechnology, including stochastic sensing.