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 was 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 P4₃₍₁₎2₁2 with cell constants of . The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of α-helical coiled-coil. Thus in the region of 10-5.5 Å resolution along the , 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 Å along the , whereas along the reflections were limited to 6.6 Å. Data analysis is under way on a data set from a PtCl₄ derivative.