Flexibility of the aldolase molecule measured using quenching-induced variations of the Forster distance for fluorescence energy transfer
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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).
KeywordsAldolase protein dynamics Forster distance fluorescence quenching
aldolase labelled with IAEDANS at Cys-289
aldolase labelled with AEDANS at Cys-289 and IAF at Cys-289
fluorescence energy transfer
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