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Biochemical analysis of a Ca 2+ ‐dependent membrane–membrane interaction mediated by the sea urchin yolk granule protein, toposome
Author(s) -
Hayley Michael,
Perera Aruni,
Robinson John J.
Publication year - 2006
Publication title -
development, growth and differentiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 66
eISSN - 1440-169X
pISSN - 0012-1592
DOI - 10.1111/j.1440-169x.2006.00872.x
Subject(s) - calcium , dissociation constant , sea urchin , biophysics , chemistry , calcium binding protein , liposome , yolk , biochemistry , membrane , granule (geology) , mole , biology , microbiology and biotechnology , receptor , paleontology , organic chemistry , food science
Toposome, a high molecular mass protein, is an abundant component of the yolk granule in the sea urchin egg and embryo. Toposome is composed of a 160 kDa polypeptide that is proteolytically processed into smaller species of 120 and 90 kDa during embryonic development. The exact biological function of toposome during early development is unknown. In this study we have examined calcium binding to toposome and the effect of this binding on the secondary and tertiary structural characteristics of the purified protein. Initially, we used equilibrium dialysis to quantify calcium binding to toposome. Monophasic binding of up to 600 M of calcium per mole of protein was detected with an intrinsic dissociation constant (calcium) of 240 µm. Increasing concentrations of calcium resulted in an increase in alpha helical content from 3.0 to 22.0%, which occurred with an apparent dissociation constant (calcium) of 25 µm. In parallel experiments, toposome binding to liposomes required similar concentrations of calcium; an apparent dissociation constant (calcium) of 25 µm was recorded. Endogenous tryptophan fluorescence measurements, both in the presence and absence of liposomes, demonstrated that the tertiary structure is sensitive to increasing concentrations of calcium with an apparent dissociation constant (calcium) of 240 µm. Toposome‐driven, liposome aggregation assays revealed a similar calcium requirement. Collectively, these results define a two‐step model for calcium modulation of toposome structure and function.