Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.     

Protein phosphatase type 1 catalytic subunit forms nondissociable dimers

Protein phosphatase type 1 is the major enzyme in skeletal muscle and liver for the dephosphorylation of Ser(P) and Thr(P) phosphoproteins. The cDNA for the catalytic subunit encodes a polypeptide of Mr 35,400 kDa, consistent with the Mr of 36,000-38,000 of the active protein purified in various laboratories. However, several investigators have found a Mr 70,000 protein for phosphatase type 1. In this report proteins of Mr 38,000 and 70,000 were resolved by Mono Q chromatography after extensive copurification from rabbit skeletal muscle. Antibodies affinity-purified against a type 1 phosphatase catalytic fragment reacted with both proteins in Western immunoblotting. Fractions from each peak were cleaved with cyanogen bromide and the major peptides were the same size by electrophoresis in gradient polyacrylamide gels. Cyanogen bromide peptides of the individual bands also were mapped by reversed-phase high-performance liquid chromatography. The purified Mr 38,000 and 70,000 proteins had identical HPLC peptide maps and also gave the same amino acid compositions after acid hydrolysis. Purified Mr 38,000 phosphatase catalytic subunit spontaneously formed a Mr 70,000 dimer that resisted usual dissociation conditions, i.e., boiling dodecyl sulfate plus 2-mercaptoethanol, but could be cleaved to about half size by various proteases, indicating that monomers were bound together near their amino or carboxy termini. Physiological changes in protein phosphatase type 1 are reflected in the amount of nondissociable dimers detected in tissue extracts.     

Biomimetic nanopores: learning from and about nature

Through recent advances in nanotechnology and molecular engineering, biomimetics - the development of synthetic systems that imitate biological structures and processes - is now emerging at the nanoscale. In this review, we explore biomimetic nanopores and nanochannels. Biological systems are full of nano-scale channels and pores that inspire us to devise artificial pores that demonstrate molecular selectivity or other functional advantages. Moreover, with a biomimetic approach, we can also study biological pores, through bottom-up engineering approaches whereby constituent components can be investigated outside the complex cellular environment.     

Chromosomal replicases as asymmetric dimers: studies of subunit arrangement and functional consequences

Studies of the DNA polymerase III holoenzyme of Escherichia coli support a model in which both the leading and lagging strand polymerases are held together in a complex with the replicative helicase and priming activities, allowing two identical alpha catalytic subunits to assume different functions on the two strands of the replication fork. Creation of distinct functions for each of the two polymerases within the holoenzyme depends on the asymmetric character of the entire complex. The asymmetry of the holoenzyme is created by the DnaX complex, a heptamer that includes tau and gamma products of the dnaX gene. tau and gamma perform unique functions in the DnaX complex, and the interaction between alpha and tau appears to dictate the catalytic subunit's role in the replicative reaction. This review considers the properties of the DnaX complex including both tau and gamma, with the goal of understanding the properties of the replicase and its function in vivo. Recent studies in eukaryotic and other prokaryotic systems suggest that an asymmetric dimeric replicase may be universal. The leading and lagging strand polymerases may be distinct in some systems. For example, Pol e and Pol delta may function as distinct leading and lagging strand polymerases in eukaryotes, and PolC and DnaE may function as distinct leading and lagging strand polymerases in low GC content Gram-positive bacteria.     

Guaiane dimers from Xylopia vielana

From the leaves of Xylopia vielana (Annonaceae) the three dimeric guaianes vielanin A-C were isolated and structurally elucidated by mass and NMR spectroscopy as 1-3. The structure of 1 contains a bridged ring system formed probably via a Diels-Alder reaction of two different guaiane monomers. Compounds 2 and 3 represent symmetric cyclobutanes formally generated from two equal guaiane moieties by [2 + 2] cycloaddition.     

Guaiane dimers from Xylopia vielana

From the leaves of Xylopia vielana (Annonaceae) two dimeric guaianes named vielanins D and E were isolated and structurally elucidated by mass and NMR spectroscopy. Vielanin D and E consist of bridged ring systems formally representing the Diels-Alder products from the hypothetical guaiane-type monomers. Due to a hemiketal function at C-8' both compounds occurred as epimeric mixtures.     

Thermal Motion of DNA in an MspA Pore

We report on an experiment and calculations that determine the thermal motion of a voltage-clamped single-stranded DNA-NeutrAvidin complex in a Mycobacterium smegmatis porin A nanopore. The electric force and diffusion constant of DNA inside a Mycobacterium smegmatis porin A pore were determined to evaluate the thermal position fluctuations of DNA. We show that an out-of-equilibrium state returns to equilibrium so quickly that experiments usually measure a weighted average over the equilibrium position distribution. Averaging over the equilibrium position distribution is consistent with results of state-of-the-art nanopore sequencing experiments. It is shown how a reduction in thermal position fluctuations can be achieved by increasing the electrophoretic force used in nanopore sequencing devices.     

Nucleotide discrimination with DNA immobilized in the MspA nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.     

Local response in nanopores

In this work the transient time correlation function (TTCF) algorithm is applied to study highly confined molecular fluids. We focus on linear polymer chains of various lengths trapped in a slab pore which is a few nanometres thick and made of atomistic walls, and the behaviour and response of the polymer melt subject to shear flow are considered. The shearing is produced by shifting the walls in opposite directions, and the temperature inside the channel is controlled by a thermostat applied to the wall atoms alone, so as to mimic the dissipation of heat as it occurs in real devices. It is shown how the TTCF algorithm can be applied to extract the fluid's dynamical and structural properties as they evolve from equilibrium and until a steady state has been established. We note that this procedure is applicable to fluids of any complexity and down to extremely low fields, comparable to those present in experimental devices. It is also shown that this technique can be used to probe local properties at specific locations across the channel. This feature is of particular significance because liquid properties inside nanoconfined geometries are mostly determined by the interactions at the interface and specifically by the structural reordering which affects the first few atomic/molecular layers close to the wall surface, e.g. slip.     

Naphthoquinone dimers and trimers from Euclea natalensis

The naphthoquinone dimers natalenone, 8′-hydroxydiospyrin and euclanone and the trimers galpinone and a compound with MW 562 were isolated from E. natalensis roots. Natalenone is a dehydrodimer of 7-methyl- juglone with the two moieties linked by two CC bonds to give a fused tetracyclic structure, one ring bearing a methylene bridge. Galpinone is a 7-methyljuglone linear trimer, the three units probably being linked C-8-C-6′and C-3′-C-3″. Euclanone is a new dimer of 7-methyljuglone and methylnaphthazarin, isomeric with 8′-hydroxydiospyrin.     

Slip boundaries in nanopores

The flow of liquid water confined within slit pores formed from amorphous surfaces of carbon and polydimethylsiloxane (PDMS) has been investigated using molecular dynamics techniques. For PDMS (including plasma treated forms) the slip length is found to be very small, and there are essentially stick boundary conditions. The slip lengths for amorphous carbon surfaces are significant; however, they are much smaller than those measured experimentally for nanopipes. We show, for the first time, that the slip length can be predicted using the Sokhan–Quirke equation for amorphous surfaces using only equilibrium properties.     

Cation transport in nanopores

Using periodic boundary conditions and external electric potential field, we have simulated an ion current flow through a flexible nanopore using cations and an explicit extended simple point charge (SPC/E) water with molecular dynamics simulation. The simulation voltages range goes beyond the usual ionic channel measurements ( ± 1 V) and yields useful information about density profiles, current density distribution and current–voltage (I–V) characteristics.