Abstract
cAMP-dependent Protein Kinase (PKA) is a protein that plays a critical role in cell signaling by means of protein phosphorylation. In this work, the kinetics of PKA activity were monitored with the aid of a single-walled carbon nanotube transistor. Molecule-by-molecule processivity was directly recorded using an individual PKA catalytic subunit attached to this sensitive transducer. The binding of adenosine triphosphate (ATP) and/or kemptide, a peptide substrate, both drove conformational changes that could be electrically monitored with sub-millisecond resolution over durations of 10 minutes or more. In environments containing either binding partner, binding and dissociation rates and rate variability were determined from many thousands of individual events. For ATP, the on-rate varies over a range of 20-1000 Hz due to dynamic disorder of the apoenzyme. In fact, single molecule monitoring allowed us to directly record periods of fast and slow binding. In the presence of both ATP and kemptide, more complicated signals resulted from PKA alternating between its apo, binary, and catalytically-active ternary complex. This three-state system was extensively monitored to determine its transition probability matrix and rates. Within our resolution of 0.1ms, the apoenzyme preferentially forms the ternary-complex, with only 27% of binding events pausing at the intermediate, binary complex. Our single molecule measurements observed a varying catalytic turnover rate of 5-100 Hz over 10 sec intervals with a time averaged rate of 30 Hz, consistent with the 20 Hz rate from ensemble measurements.
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