Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Thursday Speaker Abstracts

Subangstrom Single-Molecule Measurements of Motor Proteins Using a Nanopore Jens H. Gundlach . University of Washington, Seattle, WA, USA. We have developed a high-resolution nanopore sensor to study enzyme activity with unprecedented positional and temporal sensitivity. In this new method, single stranded DNA (or RNA) that is bound to an enzyme is drawn into the nanopore by an applied electrostatic potential. The single stranded DNA passes through the pore’s constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. The pore we use is an engineered version of the protein pore MspA in which nucleotides of the DNA strongly affect the ion current that flows through the pore’s constriction. Analysis of this ion current indicates the precise position of the DNA and thereby provides a real-time record of the enzyme’s activity. The motion of DNA can be measured on millisecond time scales with a position resolution as small as ~40 picometers, while simultaneously providing the DNA’s sequence within the enzyme. We demonstrate the extraordinary potential of this new single molecule technique on a Hel308 helicase, where we observe two distinct sub-states for each nucleotide processed. One of these about half-nucleotide long steps is ATP-dependent and the other is ATP-independent. The spatial and temporal resolution of this low-cost single molecule technique allows exploration of hitherto unobservable enzyme dynamics in real-time.

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