Single Molecule Circuits for Investigating Real-Time Binding and Enzymatic Catalysis

Single-walled carbon nanotube-based circuits, developed by the Weiss and Collins laboratories (UCI), allows for the detection of minute changes in circuit conductance caused by attached single molecules. Chemical reactions involving the attached biomolecule and the surrounding environment can be monitored in real-time.  This approach is a new method to investigate enzyme catalysis and obviates limitations associated with other single molecule techniques (e.g. photobleaching inherent to FRET). Data generated by lysozyme-functionalized nanocircuits identifies electronic signal purturbations as signatures of enzyme catalysis. These signals have been correlated to individual catalytic events: substrate binding, hydrolysis, and release. The electronic measurements distinguish seven independent time scales from a single molecule, including periods of chemically ineffective lysozyme hinge motion at 330 Hz and processive enzymatic turnover occurring around 15 Hz. Employing this novel platform for single molecule studies allowed for identification of lysozyme as a processive enzyme. In addition, lysozyme has been interrogated using natural, cross-linked peptidoglycan substrate as well as chemically-synthesized linear peptidoglycan. Lysozyme-functionalized nanocircuits exhibit slow (20 -50 Hz) and fast (200 – 400 Hz) turnover rates. However, the non-productive binding events occupy 43% of the enzyme’s time in the presence of cross-linked peptidoglycan but only 7% in the presence of linear substrate. This data provides new insight into the mechanism of T4 lysozyme. Once this new technique has been calibrated using catalysis by lysozyme, we will apply the approach to other systems. For example, caveolin-1, a protein required for formation of plasma membrane invaginations called caveolae, will be further characterized with single molecule circuits to examine its binding and inhibitory activities. Single-molecule circuits will be used to determine how and why mutations to caveolin-1 are associated with oncogenesis.