Fluorescence anisotropy as a probe to study tracer proteins in crowded solutions.
Fluorescence anisotropy has been widely used to study the dynamics and interactions of biomolecules in diluted solutions. Comparable studies on single tracer macromolecules at the cellular level are now feasible because of the recent development of non-invasive fluorescence markers, like the growing family of the green fluorescence proteins (GFPs), and the advances in time-resolved fluorescence microscopy instrumentation. The interpretation of fluorescence polarization data in terms of dynamics and biological function of the macromolecular complexes in these physiological environments requires a deep understanding of the tracer rotational diffusion in such complex media. In this work we have studied the rotational diffusion of a tracer protein, apomyoglobin labeled with 1-anilino-8-naphthalene sulfonate, in crowded solutions of an unrelated protein, ribonuclease A. We have evaluated the deviation of the different tracer rotational motions from the Stokes-Einstein-Debye diffusion behavior, and its relation to the properties of the transient molecular cavities where the tracer is rotating in the fluorescence lifetime window. Finally, we have analyzed the application of fluorescence polarization methods to determine the apparent equilibrium constants of homo and hetero-associations of macromolecules in crowded conditions.
Zorrilla
Silvia
S
Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Rivas
Germán
G
Lillo
M Pilar
MP
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D014867:Water / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D005456:Fluorescent Dyes; D009211:Myoglobin; D012996:Solutions; C010857:apomyoglobin; D014867:Water; C027132:1-anilino-8-naphthalenesulfonate; D012259:Ribonuclease, Pancreatic