Abstract
The physical structure of villin, a Ca2+-modulated regulator protein of actin filament organization, has been studied in the absence and presence of Ca2+ using analytical ultracentrifugation, gel chromatography, ultraviolet difference spectroscopy, and circular dichroism. Villin exhibits an intrinsic sedimentation coefficient, S020, w, of 5.0 s and an apparent Stokes radius, Rs, of 44 A in EGTA-containing buffer. In the presence of greater than 20 microM Ca2+, villin shows a S020, w of 4.1 s and Rs of 49 A, indicative of a conformational change in the protein. No significant changes in the partial specific volume (0.73) of villin are observed in the presence of Ca2+, and sedimentation equilibrium studies demonstrates that the effects of Ca2+ are not due to a change in the molecular mass (95,000 daltons). The combined hydrodynamic data suggest that villin is an asymmetric molecule with an axial ratio of 4.5:1, based on a prolate ellipsoid model at 0.5 g/g of hydration, corresponding to a maximum length of 84 A. The presence of Ca2+ changes the shape to a more asymmetric molecule with an axial ratio of 8:1 and a maximum length of 123 A. Since the large proteolytic fragment, villin core, does not exhibit the strong structural change of villin, an important function of the small villin headpiece domain in the observed conformational change of intact villin is suggested. The results indicate new aspects of the function of villin as cross-linker in microvillus core filament bundles and the disintegration of these structures in the presence of calcium.
Highlights
Regulator protein of actin filament organization, has The small headpiece (8500 daltonsf is a ligand-independent been studiedin the absence and presencoef Ca2+ using binding proteinand the large core (88,000 daltons)retains analytical ultracentrifugationg,el chromatography,ul- vain's calcium-dependent fiiament length restriction based traviolet difference spectroscopy, and circular dichro- on enhanced nucleation and probably direct severing
(0.73)of villin are observed in the presenocfeCa2+a, nd sedimentation equilibrium studies demonstrates that the effects of Ca2+arenotdue to achangeinthe in villin, very little is known concerning the structure of the molecule
The results indicate new aspects of the A (96 mM NaC1, 8 m~ KH2P04,5.6 mM NaZHPO.,1.5 m~ KCI, 10 functionof villin as cross-linker in microvilluscore m EDTA, 0.02% NaN3, pH 6.8) andstirredon ice for 1 h
Summary
Calcium-inducedDifference Spectra of Villin-UV difference spectroscopy on villin revealeda calcium-dependent change in the environment of the aromatic amino acids. We have confirmed the Svalue in1mM EGTA but noticed a drop to4.6 s in the presence of 1mM Ca2' This observation suggests a Ca'+-induced conformational change in villin, which was analyzed baynalytical gel chromatography at 4 "cusing a calibrated SephacrySl -300column equilibrated in 50 m~ Tris, pH 8.0, 100 m~ NaC1, 100 PM dithiothreitol, andeither 100 PM EGTAor 100 PM CaC12. The partial specific volume, 17.2,gave values of 0.737 f 0.005 and 0.733 f 0.005 in the presence of EGTA and Ca2+r,espectively These results suggest that Ca2+ binding induces only slight changes in the hydration of the protein and indicates instead that the observed Ca'+-induced changes may be due. Calcium-induced HydrodynamicChanges of Villin-In order todistinguish between a Ca2'-induced polymerization and a Ca"-induced conformational change in villin, sedimentation equilibrium was used to determine thepolymeric state of the protein.
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