Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin,vas prepared by removing 11 amino acids at the C-terminus, The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations, By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals, The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart, For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3(1))2(1)2 with cell constants of a = b = 105.6 Angstrom, c = 506.9 Angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil, Thus in the region of 10-5.5 Angstrom resolution along the c*-axis, the reflections were weak, For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background, There the crystals diffracted to beyond 3.0 Angstrom along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 Angstrom. Data analysis is under way on a data set from a PtCl4 derivative.