Brad L. Corso

Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit

Observing Protein Dynamics Following the dynamics of protein conformational changes over the relatively long periods of time typical of enzyme kinetics can be challenging. Choi et al. (p. 319; see the Perspective by Lu) were able to observe changes in lysozyme conformation, which changes its electrostatic potential, by using a carbon-nanotube field-effect transistor. Slower hydrolysis steps were compared with faster, but unproductive, hinge motion, and changes in lysozyme activity that occur with pH were shown to arise from differences in the relative amount of time spent in processive versus nonprocessive states. Changes in protein conformation can be detected via changes in electrostatic potential with a carbon nanotube transistor. Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme’s activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.

Dissecting Single-Molecule Signal Transduction in Carbon Nanotube Circuits with Protein Engineering

Single-molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, nonfluorescent single-molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems.

Electronic Measurements of Single-Molecule Catalysis by cAMP- Dependent Protein Kinase A

Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molecule of the catalytic domain of cAMP-dependent protein kinase A (PKA) was attached to a single-walled carbon nanotube device for long-duration monitoring. The electronic recording clearly resolves substrate binding, ATP binding, and cooperative formation of PKAs catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 min and tens of thousands of binding events, we determine the full transition probability matrix and conversion rates governing formation of the apo, intermediate, and closed enzyme configurations. We also observe kinetic rates varying over 2 orders of magnitude from one second to another. Anti-correlation of the on and off rates for PKA binding to the peptide substrate, but not ATP, demonstrates that regulation of enzyme activity results from altering the stability of the PKA-substrate complex, not its binding to ATP. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute useful for an enzyme with crucial roles in cell signaling.

Single Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-linked Peptidoglycan Substrates

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzymes time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.

Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment).

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzymes open and closed conformations. Consistent with these nanocircuit-based observations, the enzymes closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzymes open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Scanning Gate Spectroscopy and Its Application to Carbon Nanotube Defects

A variation of scanning gate microscopy (SGM) is demonstrated in which this imaging mode is extended into an electrostatic spectroscopy. Continuous variation of the SGM probes electrostatic potential is used to directly resolve the energy spectrum of localized electronic scattering in functioning, molecular scale devices. The technique is applied to the energy-dependent carrier scattering that occurs at defect sites in carbon nanotube transistors, and fitting energy-resolved experimental data to a simple transmission model determines the electronic character of each defect site. For example, a phenolic type of covalent defect is revealed to produce a tunnel barrier 0.1 eV high and 0.5 nm wide.

Quantitative Kelvin probe force microscopy of current-carrying devices

Kelvin probe force microscopy (KPFM) should be a key tool for characterizing the device physics of nanoscale electronics because it can directly image electrostatic potentials. In practice, though, distant connective electrodes interfere with accurate KPFM potential measurements and compromise its applicability. A parameterized KPFM technique described here determines these influences empirically during imaging, so that accurate potential profiles can be deduced from arbitrary device geometries without additional modeling. The technique is demonstrated on current-carrying single-walled carbon nanotubes (SWNTs), directly resolving average resistances per unit length of 70 kΩ/μm in semimetallic SWNTs and 200 kΩ/μm in semiconducting SWNTs.

Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology

Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozymes opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzymes state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozymes two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzymes state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.

One-dimensional Poole-Frenkel conduction in the single defect limit

A single point defect surrounded on either side by quasi-ballistic, semimetallic carbon nanotube is a nearly ideal system for investigating disorder in one-dimensional (1D) conductors and comparing experiment to theory. Here, individual single-walled nanotubes (SWNTs) are investigated before and after the incorporation of single point defects. Transport and local Kelvin Probe force microscopy independently demonstrate high-resistance depletion regions over 1.0 μm wide surrounding one point defect in semimetallic SWNTs. Transport measurements show that conductance through such wide depletion regions occurs via a modified, 1D version of Poole-Frenkel field-assisted emission. Given the breadth of theory dedicated to the possible effects of disorder in 1D systems, it is surprising that a Poole-Frenkel mechanism appears to describe defect scattering and resistance in this semimetallic system.

Electrode Characteristics of Individual, MnO2 Coated Carbon Nanotubes

Electrode Characteristics of Individual, MnO2 Coated Carbon Nanotubes Brad L. Corso, Israel Perez, and Philip G. Collins Department of Physics and Astronomy, University of California Irvine, Irvine CA 92697 We investigate interfacial charge transfer between a manganese oxide (MnO 2 ) pseudocapacitor material and graphitic carbon supports in the limit where the graphitic carbon is defect free. We achieve this limit experimentally by fabricating model electrodes comprising MnO 2 deposited on single, pristine, isolated single- walled carbon nanotubes. Li ion cyclic voltammetry of the composites gives a specific capacitance in accord with MnO 2 storage capacities, but with kinetics limited by the poor electron transfer properties of defect free carbon. By fitting the data to an equivalent circuit model, we determine the charge transfer resistivity of MnO 2 -carbon interfaces to be 9 x 10 7 Ω-cm when defects are absent, a limiting value for high power cathodes. 1. Introduction Graphitic carbons and manganese oxide (MnO 2 ) are both promising materials for heterogeneous, nanostructured pseudocapacitors because of the synergies between graphite’s high conductance and stability and MnO 2 ’s low cost and high theoretical specific capacitance (1, 2). However, existing carbon-MnO 2 composites do not achieve the full potential of both materials. This paper investigates some of the interfacial properties that can play limiting roles. Specifically, we consider the fundamental charge transfer resistance across the carbon-MnO 2 interface, and the role of carbon defect sites in reducing that resistance. It is well established that defect sites, especially those which contain oxygen, promote more efficient electron transfer by carbon electrodes (3-6). However a quantitative measure of the defect-free case has been historically elusive. High quality carbon systems all contain defects, whether at basal plane edges, grain boundaries, or points. As a result, all graphitic electrodes are ensemble mixtures of electrochemically active defects among basal plane carbon. As described in the recent review article by McCreery (3), precise control over the concentration and chemistry of defects is necessary before the fundamental electron transfer rates of these sites can be established quantitatively. To address this issue, we investigate electrochemical processes on the sidewall of a single carbon nanotube. Using high quality, single-walled carbon nanotubes (SWNTs) that are grown in place without further processing or manipulation, we interrogate carbon electrodes in the limit of defect-free carbon. Our SWNTs are electrically connected in a field effect transistor (FET) geometry, so that they can be used as both electrochemical working electrodes and as FET devices. SWNTs have an electrical conductivity that is particularly sensitive to the presence of defects (7), and this property can be exploited to characterize defects with single site sensitivity (8, 9).