Preliminary crystallographic study of turkey gizzard vinculin

Vinculin is a 117 kDa microfilament-associated protein located at the cytop lasmic aspects of focal contacts and cell-cell adherens type junctions. In both sites, vinculin participates in the formation of a submembrane 'plaque ' structure which is responsible for the attachment of actin filaments to t he plasma membrane. Vinculin consists of 1066 amino acids, which form a lar ge 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two d omains are separated by several stretches of proline residues where the maj or proteolytic cleavage sites are located. The experimental procedure for i solation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obta ined. The homogeneity of the vinculin solution was analyzed prior to crysta llization using dynamic light scattering. Crystals of vinculin have been ob tained in buffer containing 2 mg ml(-1) protein, 0.9 M ammonium sulfate, 0. 1 M MES pH 6.5 using both the hanging-drop and sitting-drop vapour-diffusio n methods. The crystals have the form of rhombic plates and grow to maximal dimensions of 0.3 x 0.3 x 0.05 mm in two weeks. Preliminary X-ray data sho w that the crystals diffract to 3.5 Angstrom resolution at the X11 beamline of DESY and belong to the monoclinic space group P2(1). Crystal unit-cell parameters are estimated to be a = 57, b = 351, c = 70 Angstrom, alpha = 90 , beta = 113, gamma = 90 degrees.