O. Tolga Gul

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.

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.

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.

Single molecule sensing with carbon nanotube devices

Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes have the requisite sensitivity to detect single molecule events and sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. In each case, recordings resolved detailed trajectories of tens of thousands of individual chemical events and provided excellent statistics for single-molecule events. This electronic technique has a temporal resolution approaching 1 microsecond, which provides a new window for observing brief, intermediate transition states. In addition, the devices are indefinitely stable, so that the same molecule can be observed for minutes and hours. The extended recordings provide new insights into rare events like transitions to chemically-inactive conformations.

Electronic Effects of Defects in One-Dimensional Channels

As electronic devices shrink to the one-dimensional limit, unusual device physics can result, even at room temperature. Nanoscale conductors like single-walled carbon nanotubes (SWNTs) are particularly useful tools for experimentally investigating these effects. Our characterization of point defects in SWNTs has focused on these electronic consequences. A single scattering site in an otherwise quasi-ballistic SWNT introduces resistance, transconductance, and chemical sensitivity, and here we investigate these contributions using a combination of transport and scanning probe techniques. The transport measurements determine the two-terminal contributions over a wide range of bias, temperature, and environmental conditions, while the scanning probe work provides complementary confirmation that the effects originate at a particular site along the conduction path in a SWNT. Together, the combination proves that single point defects behave like scattering barriers having Poole-Frenkel transport characteristics. The Poole-Frenkel barriers have heights of 10 – 30 meV and gate-dependent widths that grow as large as 1 μm due to the uniquely poor screening in one dimension. Poole-Frenkel characteristics suggest that the barriers contain at least one localized electronic state, and that this state primarily contributes to conduction under high bias or high temperature conditions. Because these localized states vary from one device to another, we hypothesize that each might be unique to a particular defect’s chemical type.