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.