Single molecule processivity and dynamics of cAMP-dependent protein kinase (PKA)

ORAL

Abstract

Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 $\mu $s resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Statistical analysis of many events determines on- and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.

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Authors

  • Patrick C. Sims

    • Department of Physics and Astronomy, University of California, Irvine

  • Yongki Choi

    • Department of Physics and Astronomy, University of California, Irvine

  • Chengjun Dong

    • Department of Physics and Astronomy, University of California, Irvine

  • Issa S. Moody

    • Department of Molecular Biology and Biochemistry, University of California, Irvine

  • Mariam Iftikhar

    • Department of Chemistry, University of California, Irvine

  • O. Tolga Gul

    • Department of Physics and Astronomy, University of California, Irvine

  • Gregory A. Weiss

    • Department of Molecular Biology, University of California, Irvine
    • Departments of Molecular Biology and Biochemistry, and Chemistry, University of California, Irvine

  • Phillip Collins

    • Department of Physics and Astronomy, University of California, Irvine
    • University of California, Irvine
    • University of California Irvine
    • University of California, Riverside