Fretting about FRET: Correlation between κ and R

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 〈κ2〉 is found to deviate significantly from the theoretical value and high correlation between the orientation factor κ 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 κ and R and as high as 0.68 between κ2 and R. The presence of this correlation highlights the fact that essentially all fluorescence-detected resonance energy transfer studies have assumed that κ and R are independent—an assumption that is clearly not justified in the system studied here. The correlation results in the quantities 〈κ2R−6〉 and 〈κ2〉 〈R−6〉 differing by a factor of 1.6. The observed correlation between κ and R is caused by the succinimide linkage between the D and HEWL, which is found to be relatively inflexible.