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Flexibility of the aldolase molecule measured using quenching-induced variations of the Forster distance for fluorescence energy transfer

  • Piotr Dobryszycki
  • Dorota Krzyzanowska
  • Marian Kochman
Special Issue on Lasers in Chemistry, Proceedings of the Conference of the International Centre for Pure and Applied Chemistry, Trieste, June 1990
  • 13 Downloads

Abstract

The range of flexibility of the rabbit muscle aldolase molecule was studied using fluorescent labelled aldolase. The protein molecule was specifically labelled on the opposite sites of the enzyme subunit with the fluorescence energy donor and acceptor residues. Labelled aldolase with full enzymatic activity was used as a tool in the FRET studies between IAEDANS — donor or Cys-289 and IAF — acceptor on Cys-239. A range of Forster distance (R) was obtained by collisional quenching of the donor emission. The experiments of donor fluorescence quenching with wide range of acrylamide concentrations have shown the changes of donor-acceptor distances. In the absence of quencher the D-A distance distribution in characterized by an average value of 40.4 Å, and a half-width of 0.13 Å. A dramatic increase in half-width to 17.7Å is observed after expositions of the enzyme to high acrylamide concentrations (0.13 M-0.68 M).

Keywords

Aldolase protein dynamics Forster distance fluorescence quenching 

Abbreviations

IAEDANS

5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulphonic acid

IAF

5-iodoacetamidofluorescein

aldolase-AEDANS

aldolase labelled with IAEDANS at Cys-289

aldolase-AEDANS-IAF

aldolase labelled with AEDANS at Cys-289 and IAF at Cys-289

FRET

fluorescence energy transfer

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Copyright information

© Indian Academy of Sciences 1991

Authors and Affiliations

  • Piotr Dobryszycki
    • 1
  • Dorota Krzyzanowska
    • 1
  • Marian Kochman
    • 1
  1. 1.Department of Biochemistry, Institute for Organic Physical ChemistryTechnical UniversityWroclawPoland

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