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Understanding Single-Molecule Protein Dynamics via Electronic Circuit

  • Author(s): Iftikhar, Mariam
  • Advisor(s): Weiss, Gregory A
  • et al.
Abstract

Molecular motions of proteins and their flexibility induce 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 millisecond to microsecond range where conventional optical techniques and computational modeling are most limited. To investigate the microsecond dynamics of enzymes, single-walled carbon nanotube – field-effect transistors (SWCNT-FETs) were used as single-molecule biosensors. The SWCNT-FETs have sufficient sensitivity and bandwidth to monitor the conformational motions and processivity of an individual cAMP-dependent protein kinase A (PKA) molecule. Protein attachment is accomplished by functionalizing a SWCNT-FET device with a single protein and measuring the conductance versus time through the device as it is submerged in an electrolyte solution. PKA-functionalized nanocircuits elucidated the time PKA spent traveling between three different conformational motions upon binding of its two substrates.

To generalize this approach for the study of a wide variety of proteins at the single-molecule level, this dissertation investigates the bioconjugation process to determine and isolate the key parameters required for functionalizing a SWCNT with a single protein. Further analysis into the parameters implicated in the thiol-maleimide bioconjugation step of the SWCNT-FET device fabrication proved an exhausting effort. We developed improved linker molecules, anthracene maleimide, for attachment of three different proteins to the SWCNT and a new derivative of maleimide providing a solution to overcome side reactions associated with the reducing agent, tris(2-carboxyethyl)phosphine (TCEP). Protein purity and presence of detergent proved to play key roles in preventing nanocircuit surface fouling. An alternative bioconjugation method, utilizing an azide-functionalized hen egg-white lysozyme (HEWL) to tether the enzyme to the SWCNT through an alkyne linker molecule was developed. The work described here explores the current SWCNT attachment method as well as a new strategy to apply SWCNT-FET devices to study novel proteins. The results demonstrate SWCNT-FET as a sensitive technique for studying conformational dynamics of biological molecules in the microsecond range.

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