Fretting about FRET: correlation between kappa and R

Biophys J. 2007 Jun 15;92(12):4168-78. doi: 10.1529/biophysj.106.092650. Epub 2007 Mar 23.

Abstract

Molecular dynamics simulations were used to examine the structural dynamics of two fluorescent probes attached to a typical protein, hen egg-white lysozyme (HEWL). The donor probe (D) was attached via a succinimide group, consistent with the commonly-used maleimide conjugation chemistry, and the acceptor probe (A) was bound into the protein as occurs naturally for HEWL and the dye Eosin Y. The <kappa(2)> is found to deviate significantly from the theoretical value and high correlation between the orientation factor kappa and the distance R is observed. The correlation is quantified using several possible fixed A orientations and correlation as high as 0.80 is found between kappa and R and as high as 0.68 between kappa(2) and R. The presence of this correlation highlights the fact that essentially all fluorescence-detected resonance energy transfer studies have assumed that kappa and R are independent--an assumption that is clearly not justified in the system studied here. The correlation results in the quantities <kappa(2)R(-)(6)> and <kappa(2)> < R(-)(6)> differing by a factor of 1.6. The observed correlation between kappa and R is caused by the succinimide linkage between the D and HEWL, which is found to be relatively inflexible.

Publication types

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

MeSH terms

  • Binding Sites
  • Computer Simulation
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry*
  • Models, Chemical*
  • Models, Molecular*
  • Muramidase / chemistry*
  • Muramidase / ultrastructure*
  • Protein Binding
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Statistics as Topic

Substances

  • Fluorescent Dyes
  • Muramidase