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Microsecond Dynamics of Enzymes: A Single-Molecule Study Using Carbon Nanotube Transistors

  • Author(s): Akhterov, Maxim V.
  • Advisor(s): Collins, Philip G.
  • et al.
Abstract

Molecular motions of proteins and their flexibility determine conformational states required for enzyme catalysis, signal transduction, and protein-protein interactions. However, the mechanisms for protein transitions between conformational states are often poorly understood, especially in the milli- to microsecond ranges where conventional optical techniques and computational modeling are most limited. The goal of this work was to use single-walled carbon nanotube field-effect transistors (SWCNT-FET) as single-molecule biosensors to investigate microsecond dynamics of three enzymes. Low-noise electrical transport measurements were performed in a home-built electrochemical flow cell. To ensure that the output signal was free from external noise across a 200 kHz measurement bandwidth, parasitic capacitance was minimized by tightly integrating electrical components and constraining the liquid within a microfluidic channel. After attaching a single enzyme molecule to the SWCNT sidewall, dynamic changes of conductance through a SWCNT-FET reported conformational motions of an enzyme with a 1.6 $\mu$s resolution. This technique was used to study microsecond dynamics of T4 Lysozyme, Klenow Fragment of DNA polymerase I (KF), and protein kinase A (PKA).

Lysozyme closing and opening took on average 37 $\mu$s. The distribution of transition durations was independent of the lysozyme state: either catalytic or nonproductive. The observed symmetry in enzyme opening and closing suggests that substrate translocation occurs while lysozyme is closed. For KF and PKA, the microsecond resolution revealed that the transition duration was 3-5 $\mu$s for both enzymes, about ten times shorter than lysozyme. Additionally, the high-bandwidth recording of KF resolved fast non-catalytic closures. When KF was processing poly(dT)$_{42}$ template KF stayed 2-3 times longer in a closed conformation than when it was replicating poly(dA)$_{42}$. However, the open events of KF were 2-5 times shorter when it was processing poly(dT)$_{42}$ compared to poly(dA)$_{42}$. The results demonstrate SWCNT-FET as a sensitive technique for studying conformational dynamics of biological molecules in the microsecond range.

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