Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/11721
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Título : Protein self-association in crowded protein solutions: a time-resolved fluorescence polarization study
Autor : Zorrilla, Silvia, Rivas, Germán, Acuña, A. Ulises, Lillo, M. Pilar
Palabras clave : Time-resolved fluorescence anisotropy
Macromolecular crowding
Self-association
Segmental flexibility
Apomyoglobin
Ribonuclease A
Human serum albumin
Fecha de publicación : 2004
Editor: John Wiley & Sons
Citación : Protein Science 13: 1-10 (2004)
Resumen: The self-association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8-anilinonaphthalene-1-sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50-200 mg/mL range, using picosecond-resolved fluorescence anisotropy methods. It was found that apoMb molecules self-associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time-resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.
Versión del editor: http://dx.doi.org/10.1110/ps.04809404
URI : http://hdl.handle.net/10261/11721
ISSN: 0961-8368
DOI: 10.1110/ps.04809404
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