Fretting about FRET: Correlation between [kappa] and R

• Published in: Biophysical journal 2007-06, Vol.92 (12), p.4168
• Main Authors: VanBeek, Darren B, Zwier, Matthew C, Shorb, Justin M, Krueger, Brent P
• Format: Article
• Language: English
• Subjects: Biochemistry; Fluorescence; Molecular biology; Proteins
• ISSN: 0006-3495; 1542-0086

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 malelmide conjugation chemistry, and the acceptor probe (A) was bound into the protein as occurs naturally for HEWL and the dye Eosin Y. The (κ^sup 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^sup 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 [left angle bracket]κ^sub 2^R^sup -6^[right angle bracket] and [left angle bracket]κ^sup 2^[right angle bracket] [left angle bracket]R^sup -6^[right angle bracket] 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. [PUBLICATION ABSTRACT]