Fast Force Clamp in Optical Tweezers: A Tool to Study the Kinetics of Molecular Reactions

  • Pasquale Bianco
  • Lorenzo Bongini
  • Luca Melli
  • Giulia Falorsi
  • Luca Salvi
  • Dan Cojoc
  • Vincenzo Lombardi
Conference paper
Part of the Advances in Atom and Single Molecule Machines book series (AASMM)

Abstract

A dual-laser optical tweezers has been developed to study the mechanics of motor proteins or DNA filaments. A bead attached to one end of the specimen is trapped in the confocal point of the two lasers, while the other end is connected to a three-dimensional piezo-stage. The instrument can be operated under computer control either as a length clamp, applying length steps or ramps, or as a force clamp, applying abrupt changes in load of fixed magnitude and direction. The dynamic range of the instrument (0.5–75,000 nm in length and 0.5–200 pN in force) and the speed of the force feedback permit recording the kinetics of molecular and intermolecular phenomena such as the overstretching transition in double-stranded DNA (ds-DNA) or the generation of force and shortening by an ensemble of myosin motors pulling on an actin filament. We demonstrate the performance of the system in recording for the first time the transient kinetics of the ds-DNA overstretching transition, which allows the determination of the underlying reaction parameters, such as rate constants and distance to the transitions state.

Keywords

Polystyrene Barb 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Pasquale Bianco
    • 1
  • Lorenzo Bongini
    • 1
  • Luca Melli
    • 1
  • Giulia Falorsi
    • 1
  • Luca Salvi
    • 1
  • Dan Cojoc
    • 2
  • Vincenzo Lombardi
    • 1
  1. 1.Laboratory of Physiology, Department of BiologyUniversity of FlorenceFlorenceItaly
  2. 2.IOM—National Research CouncilTriesteItaly

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