Fretting about FRET: Correlation between κ and R

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 〈 κ 2〉 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 κ 2 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 〈 κ 2 R −6 〉 and 〈 κ 2〉 〈 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.