Tethering a single lysozyme molecule to a carbon nanotube field effect transistor (FET) 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 we re 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.

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, non-fluorescent 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.

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 enzyme’s open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme 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 enzyme open state, but with a nearly two-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.

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 exhibits processive slow turnover rates of 20 – 50 s−1 and rapid 200 – 400 s−1 non-productive motions. The latter, nonproductive binding events occupy 43% of the enzyme’s 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 appears to sidestep the peptide cross-links to zigzag through the wild-type substrate.

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 (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 PKA’s catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 minutes 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 two 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.