Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric alpha-hemolysin pore (alpha HL) has been engineered to form a nanoreactor to study covalent chemistry at the single -molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different alpha HL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic alpha HL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single -molecule level

Unzipping Kinetics of Double-Stranded DNA in a Nanopore

We studied the unzipping kinetics of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. PCR analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.Comment: 10 pages, 5 figures, Accepted: Physics Review Letter

The NTD Nanoscope: potential applications and implementations

<p>Abstract</p> <p>Background</p> <p>Nanopore transduction detection (NTD) offers prospects for a number of highly sensitive and discriminative applications, including: (i) single nucleotide polymorphism (SNP) detection; (ii) targeted DNA re-sequencing; (iii) protein isoform assaying; and (iv) biosensing via antibody or aptamer coupled molecules. Nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. The NTD Nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. Meta Logos Inc. is developing both pre-packaged device platforms and component-level (unassembled) kit platforms (the latter described here). In both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. In this paper we provide an overview of the NTD Nanoscope applications and implementations. The NTD Nanoscope Kit, in particular, is a component-level reproduction of the standard NTD device used in previous research papers.</p> <p>Results</p> <p>The NTD Nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. To expedite set-up in new lab settings, the calibration and troubleshooting for the NTD Nanoscope kit components and signal processing software, the NTD Nanoscope Kit, is designed to include a set of test buffers and control molecules based on experiments described in previous NTD papers (the model systems briefly described in what follows). The description of the Server-interfacing for advanced signal processing support is also briefly mentioned.</p> <p>Conclusions</p> <p>SNP assaying, SNP discovery, DNA sequencing and RNA-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction (PCR) enzyme. The NTD Nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead.</p

Kinetics and regulation of two catalytic subunits of cAMP-dependent protein kinase from Aplysia californica.

CAPL-A1 and CAPL-A2, two catalytic subunits of Aplysia cAMP-dependent protein kinase, are encoded by mRNAs generated by alternative splicing of transcripts of a gene that contains two mutually exclusive exon cassettes. The subunits are identical except for amino acids 142-183 of the 352 residues, which differ at 10 of 42 positions. CAPL-A1 and CAPL-A2 have now been expressed in insect cells and purified to homogeneity. The subunits differ in their catalytic properties, which have been determined with a series of synthetic peptide substrates. For example, kcat and Km values for the peptide LRRASLG (kemptide) are 42 s-1 and 36 microM and 28 s-1 and 17 microM for CAPL-A1 and CAPL-A2, respectively. CAPL-A1 and CAPL-A2 have different substrate specificities. For example, (kcat/Km)peptide-T/(kcat/Km)kemptide is 9.1 x 10(-3) for CAPL-A1 and 15 x 10(-3) for CAPL-A2, where peptide-T is the kemptide homologue LRRATLG. The subunits also differ in regulation as determined by their interactions with a purified type I regulatory subunit, which has an IC50 for CAPL-A1 that is 3.5 times higher than the IC50 for CAPL-A2. These modest differences reinforce accumulating evidence that the physiological state of a cell depends upon a spectrum of protein kinases with overlapping substrate specificities and regulatory properties

Kinetics and regulation of two catalytic subunits of cAMP-dependent protein kinase from Aplysia californica.

CAPL-A1 and CAPL-A2, two catalytic subunits of Aplysia cAMP-dependent protein kinase, are encoded by mRNAs generated by alternative splicing of transcripts of a gene that contains two mutually exclusive exon cassettes. The subunits are identical except for amino acids 142-183 of the 352 residues, which differ at 10 of 42 positions. CAPL-A1 and CAPL-A2 have now been expressed in insect cells and purified to homogeneity. The subunits differ in their catalytic properties, which have been determined with a series of synthetic peptide substrates. For example, kcat and Km values for the peptide LRRASLG (kemptide) are 42 s-1 and 36 microM and 28 s-1 and 17 microM for CAPL-A1 and CAPL-A2, respectively. CAPL-A1 and CAPL-A2 have different substrate specificities. For example, (kcat/Km)peptide-T/(kcat/Km)kemptide is 9.1 x 10(-3) for CAPL-A1 and 15 x 10(-3) for CAPL-A2, where peptide-T is the kemptide homologue LRRATLG. The subunits also differ in regulation as determined by their interactions with a purified type I regulatory subunit, which has an IC50 for CAPL-A1 that is 3.5 times higher than the IC50 for CAPL-A2. These modest differences reinforce accumulating evidence that the physiological state of a cell depends upon a spectrum of protein kinases with overlapping substrate specificities and regulatory properties

Redirecting pore assembly of staphylococcal α-hemolysin by protein engineering

α-Hemolysin (αHL), a β-barrel pore-forming toxin (βPFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, αHL assembles to form heptameric membrane-spanning pores. We have previously engineered αHL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned αHL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of αHL with galectin-1 (αHLG1, αHL-Galectin-1 chimera). αHLG1 was cytolytic toward cells that lack a receptor for wild-type αHL. We then constructed protease-activatable mutants of αHLG1 (PAMαHLG1s). PAMαHLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications

Redirecting pore assembly of staphylococcal α-hemolysin by protein engineering

α-Hemolysin (αHL), a β-barrel pore-forming toxin (βPFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, αHL assembles to form heptameric membrane-spanning pores. We have previously engineered αHL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned αHL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of αHL with galectin-1 (αHLG1, αHL-Galectin-1 chimera). αHLG1 was cytolytic toward cells that lack a receptor for wild-type αHL. We then constructed protease-activatable mutants of αHLG1 (PAMαHLG1s). PAMαHLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications