Non-linear least-squares methods applied to the analysis of fluorescence energy transfer measurements.
We describe a new method for calculating the efficiency of fluorescence energy transfer on labeled macromolecules using steady-state measurements. A single estimation of the efficiency value is obtained by a global analysis of all the measurement data sets (absorption, emission and excitation spectra) using non-linear least-squares. The method was tested on simulated and experimental data obtained from three simple model compounds: an equimolar mixture of tryptophan-tyrosine and two peptides, Trp-Tyr and Trp-Gly-Gly-Tyr, in which transfer efficiencies are respectively nearly 100% and 50%. The method was found to be reliable and provides methodological and quantitative advantages in regard to the sequential methods currently used.
Flamion
P J
PJ
Laboratoire de Biophysique, U.F.R. des Sciences Pharmaceutiques et Biologiques, Dijon, France.
Cachia
C
C
Schreiber
J P
JP
D000595:Amino Acid Sequence; D004735:Energy Transfer; D016018:Least-Squares Analysis; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence / Q000379:methods
D046911:Macromolecular Substances