Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay.
Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.
Bialik
C N
CN
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Wolf
B
B
Rachofsky
E L
EL
Ross
J B
JB
Laws
W R
WR
D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000465:Algorithms; D000818:Animals; D003198:Computer Simulation; D005454:Fluorescence Polarization; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D009010:Monte Carlo Method; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D046911:Macromolecular Substances; D014364:Tryptophan; D000426:Alcohol Dehydrogenase