Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Conversion of Serine-Type Aldehyde Tags by the Radical SAM Protein AtsB from Methanosarcina mazei.

Formylglycine-generating enzymes provide a convenient tool for site-specific protein derivatization. Their ability to oxidize cysteine or serine residues within a defined consensus sequence to Cα -formylglycine (FGly) allows for the targeted introduction of a unique chemical handle for various bioconjugation reactions. In recent years, oxygen-dependent FGly-generating enzyme saw broad use in protein functionalization and the generation of protein conjugates. Yet, the FGly-generating system AtsB, along with its capability to convert unusual aldehyde tag sequences, remains mostly unused. Herein, the ability of AtsB from Methanosarcina mazei to convert nonclassical aldehyde tags of the SX(A/P)XR-type and its potential use in bioconjugation chemistry are demonstrated.

Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I).

The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217-Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed.

Energy transducing roles of antiporter-like subunits in Escherichia coli NDH-1 with main focus on subunit NuoN (ND2).

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mß sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.

Randomly selected suppressor mutations in genes for NADH : quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain.

The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH : quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH : quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria-Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH : quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH : quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation.

Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation.

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is the first and largest enzyme of the respiratory chain which has a central role in cellular energy production and is implicated in many human neurodegenerative diseases and aging. It is believed that the peripheral domain of complex I/NDH-1 transfers the electron from NADH to Quinone (Q) and the redox energy couples the proton translocation in the membrane domain. To investigate the mechanism of the proton translocation, in a series of works we have systematically studied all membrane subunits in the Escherichia coli NDH-1 by site-directed mutagenesis. In this mini-review, we have summarized our strategy and results of the mutagenesis by depicting residues essential for proton translocation, along with those for subunit connection. It is suggested that clues to understanding the driving forces of proton translocation lie in the similarities and differences of the membrane subunits, highlighting the communication of essential charged residues among the subunits. A possible proton translocation mechanism with all membrane subunits operating in unison is described.

Distinctive solvation patterns make renal osmolytes diverse.

The kidney uses mixtures of five osmolytes to counter the stress induced by high urea and NaCl concentrations. The individual roles of most of the osmolytes are unclear, and three of the five have not yet been thermodynamically characterized. Here, we report partial molar volumes and activity coefficients of glycerophosphocholine (GPC), taurine, and myo-inositol. We derive their solvation behavior from the experimental data using Kirkwood-Buff theory. We also provide their solubility data, including solubility data for scyllo-inositol. It turns out that renal osmolytes fall into three distinct classes with respect to their solvation. Trimethyl-amines (GPC and glycine-betaine) are characterized by strong hard-sphere-like self-exclusion; urea, taurine, and myo-inositol have a tendency toward self-association; sorbitol and most other nonrenal osmolytes have a relatively constant, intermediate solvation that has components of both exclusion and association. The data presented here show that renal osmolytes are quite diverse with respect to their solvation patterns, and they can be further differentiated based on observations from experiments examining their effect on macromolecules. It is expected, based on the available surface groups, that each renal osmolyte has distinct effects on various classes of biomolecules. This likely allows the kidney to use specific combinations of osmolytes independently to fine-tune the chemical activities of several types of molecules.

Roles of subunit NuoK (ND4L) in the energy-transducing mechanism of Escherichia coli NDH-1 (NADH:quinone oxidoreductase).

The bacterial H(+)-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1-3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue ((K)Glu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue ((K)Glu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted (K)Glu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of (K)Glu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK ((K)Arg-25, (K)Arg-26, and (K)Asn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.

Electron transfer in subunit NuoI (TYKY) of Escherichia coli NADH:quinone oxidoreductase (NDH-1).

Bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of a peripheral and a membrane domain. The peripheral domain catalyzes the electron transfer from NADH to quinone through a chain of seven iron-sulfur (Fe/S) clusters. Subunit NuoI in the peripheral domain contains two [4Fe-4S] clusters (N6a and N6b) and plays a role in bridging the electron transfer from cluster N5 to the terminal cluster N2. We constructed mutants for eight individual Cys-coordinating Fe/S clusters. With the exception of C63S, all mutants had damaged architecture of NDH-1, suggesting that Cys-coordinating Fe/S clusters help maintain the NDH-1 structure. Studies of three mutants (C63S-coordinating N6a, P110A located near N6a, and P71A in the vicinity of N6b) were carried out using EPR measurement. These three mutations did not affect the EPR signals from [2Fe-2S] clusters and retained electron transfer activities. Signals at g(z) = 2.09 disappeared in C63S and P110A but not in P71A. Considering our data together with the available information, g(z,x) = 2.09, 1.88 signals are assigned to cluster N6a. It is of interest that, in terms of g(z,x) values, cluster N6a is similar to cluster N4. In addition, we investigated the residues (Ile-94 and Ile-100) that are predicted to serve as electron wires between N6a and N6b and between N6b and N2, respectively. Replacement of Ile-100 and Ile-94 with Ala/Gly did not affect the electron transfer activity significantly. It is concluded that conserved Ile-100 and Ile-94 are not essential for the electron transfer.

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli.

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.

A Scalable CMOS Molecular Electronics Chip for Single-Molecule Biosensing.

This work reports the first CMOS molecular electronics chip. It is configured as a biosensor, where the primary sensing element is a single molecule "molecular wire" consisting of a ∼100 GΩ, 25 nm long alpha-helical peptide integrated into a current monitoring circuit. The engineered peptide contains a central conjugation site for attachment of various probe molecules, such as DNA, proteins, enzymes, or antibodies, which program the biosensor to detect interactions with a specific target molecule. The current through the molecular wire under a dc applied voltage is monitored with millisecond temporal resolution. The detected signals are millisecond-scale, picoampere current pulses generated by each transient probe-target molecular interaction. Implemented in a 0.18 µm CMOS technology, 16k sensors are arrayed with a 20 µm pitch and read out at a 1 kHz frame rate. The resulting biosensor chip provides direct, real-time observation of the single-molecule interaction kinetics, unlike classical biosensors that measure ensemble averages of such events. This molecular electronics chip provides a platform for putting molecular biosensing "on-chip" to bring the power of semiconductor chips to diverse applications in biological research, diagnostics, sequencing, proteomics, drug discovery, and environmental monitoring.

Pivotal roles of three conserved carboxyl residues of the NuoC (30k) segment in the structural integrity of proton-translocating NADH-quinone oxidoreductase from Escherichia coli.

The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN or Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB, -C, -D, -E, -F, -G, and -I) and the membrane arm (NuoA, -H, -J, -K, -L, -M, and -N). In Escherichia coli NDH-1, the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor and quinone association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In this work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are thought to be in the third α-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, we propose that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in the structural stability of NDH-1.

Features of subunit NuoM (ND4) in Escherichia coli NDH-1: TOPOLOGY AND IMPLICATION OF CONSERVED GLU144 FOR COUPLING SITE 1.

The bacterial H(+)-pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA-N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I-IX and XII-XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu(144) of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914-36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu(144) for proton translocation has been discussed.

Review of Inguinal Hernia Repairs by Various Surgical Techniques in a District General Hospital in the UK.

Inguinal hernia is the commonest surgical operation. This is a large study from a district general hospital. The study spanned over 2 years with 2 further years of follow up. It is a retrospective analysis of eight hundred and seventy seven (877)inguinal hernia operations performed in a district general hospital. The following factors were looked at: type of repair, grade of surgeon performing the procedure and outcome of various repairs. The results showed that the most common technique was the Lichenstein's repair(58%). Recurrence rates were between 0.4%-30% depending on types of hernia repair.

Critical roles of subunit NuoH (ND1) in the assembly of peripheral subunits with the membrane domain of Escherichia coli NDH-1.

The bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of two domains, a peripheral arm and a membrane arm. NuoH is a counterpart of ND1, which is one of seven mitochondrially encoded hydrophobic subunits, and is considered to be involved in quinone/inhibitor binding. Sequence comparison in a wide range of species showed that NuoH is comprehensively conserved, particularly with charged residues in the cytoplasmic side loops. We have constructed 40 mutants of 27 conserved residues predicted to be in the cytoplasmic side loops of Escherichia coli NuoH by utilizing the chromosomal DNA manipulation technique and investigated roles of these residues. Mutants of Arg(37), Arg(46), Asp(63), Gly(134), Gly(145), Arg(148), Glu(220), and Glu(228) showed low deamino-NADH-K(3)Fe(CN)(6) reductase activity, undetectable NDH-1 in Blue Native gels, low contents of peripheral subunits (especially NuoB and NuoCD) bound to the membranes, and a significant loss of the membrane potential and proton-pumping function coupled to deamino-NADH oxidation. The results indicated that these conserved residues located in the cytoplasmic side loops are essential for the assembly of the peripheral subunits with the membrane arm. Implications for the involvement of NuoH (ND1) in maintaining the structure and function of NDH-1 are discussed.

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design.

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.