Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics.
Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.
Feinstein
Efraim
E
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Deikus
Gintaras
G
Rusinova
Elena
E
Rachofsky
Edward L
EL
Ross
J B Alexander
JB
Laws
William R
WR
D000818:Animals; D016880:Anisotropy; D005399:Fishes / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D008027:Light; D008958:Models, Molecular; D009038:Motion; D010320:Parvalbumins / Q000737:chemistry* / Q000145:classification / Q000528:radiation effects*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D011786:Quality Control; D051381:Rats; D011922:Rats, Mutant Strains; D015203:Reproducibility of Results; D012212:Rheology / Q000379:methods; D012399:Rotation; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D013045:Species Specificity; D014364:Tryptophan / Q000737:chemistry; D014783:Viscosity; D014867:Water / Q000737:chemistry*
D010320:Parvalbumins; D011506:Proteins; D012996:Solutions; D014867:Water; D014364:Tryptophan