Submillisecond conformational changes in proteins resolved by photothermal beam deflection

J Vis Exp. 2014 Feb 18:(84):e50969. doi: 10.3791/50969.

Abstract

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca(2+) binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (τ < 20 μsec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Video-Audio Media

MeSH terms

  • Acetates / chemistry
  • Calcium / chemistry
  • Calcium / metabolism
  • Calorimetry / methods*
  • Ethylenediamines / chemistry
  • Kinetics
  • Kv Channel-Interacting Proteins / chemistry*
  • Kv Channel-Interacting Proteins / metabolism
  • Photochemical Processes
  • Protein Conformation
  • Thermodynamics
  • Thermography / methods

Substances

  • Acetates
  • Ethylenediamines
  • Kv Channel-Interacting Proteins
  • DM-nitrophen
  • Calcium