Preliminary crystallographic study of turkey gizzard vinculin

Abstract

Vinculin is a 117 kDa microfilament-associated protein located at the cytoplasmic 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 the plasma membrane. Vinculin consists of 1066 amino acids, which form a large 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two domains are separated by several stretches of proline residues where the major proteolytic cleavage sites are located. The experimental procedure for isolation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obtained. The homogeneity of the vinculin solution was analyzed prior to crystallization using dynamic light scattering. Crystals of vinculin have been obtained 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-diffusion 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 show that the crystals diffract to 3.5 A 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 A, alpha = 90, beta = 113, gamma = 90 degrees.

Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

“…The obstacles encountered in obtaining good data sets for metavinculin-ÁLeu954 DCM mutant crystals are similar to those experienced with human (Borgon et al, 2004), chicken (Bakolitsa et al, 2004) and turkey gizzard (Kogan et al, 2000) vinculin. These hurdles were not the consequence of specific crystallization conditions: metavinculin crystals were generated in 0.6 M sodium/potassium phosphate and 175 mM ammonium sulfate, those of turkey and chicken vinculin grew in 0.9-1.2 M ammonium sulfate (Kogan et al, 2000;Bakolitsa et al, 2004) and human vinculin crystals grew in 10%(v/v) PEG 2000 (Borgon et al, 2004). Notably, metavinculin and vinculin crystals appear similar in their radiation-sensitivity and are markedly improved by streak-seeding: human vinculin streak-seeded crystallization communications Acta Cryst.…”

“…Furthermore, human metavinculin and vinculin streak-seeded crystals overcome degradation as they appeared overnight and achieved their final size shortly thereafter, compared with the 10 d crystal growth without streak-seeding. Likewise, the crystals of turkey gizzard vinculin that diffacted to 3.5 Å resolution took two weeks to attain maximum size (Kogan et al, 2000). The radiation-sensitive nature of vinculin crystals has been well documented: data sets from several isomorphic human vinculin crystals had to be merged and scaled together to obtain a complete 2.85 Å resolution data set that was suitable for structure determination (Borgon et al, 2004).…”