Fretting about FRET: correlation between kappa 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 <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.
VanBeek
Darren B
DB
Hope College, Department of Chemistry, Holland, Michigan, USA.
Zwier
Matthew C
MC
Shorb
Justin M
JM
Krueger
Brent P
BP
D001665:Binding Sites; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic
D005456:Fluorescent Dyes; D009113:Muramidase