PRODUCTION AND CRYSTALLIZATION OF LOBSTER MUSCLE TROPOMYOSIN EXPRESSED IN SF9 CELLS

A new form of muscle tropomyosin crystal has been obtained, by employi ng new strategies in protein preparation and crystallization. Non-poly merizable 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 h omogeneous protein preparations, By routinely monitoring homogeneity b y mass spectrometry, we found that the homogeneity played a key role i n obtaining good crystals, The crystal quality was also dependent on i soforms; the crystals raised from a slow muscle-specific isoform diffr acted to a higher resolution, compared with a fast muscle-specific cou nterpart, 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 c ell constants of a = b = 105.6 Angstrom, c = 506.9 Angstrom. The cryst als gave rise to reflections the intensities of which were characteris tically determined by the transform of alpha-helical coiled-coil, Thus in the region of 10-5.5 Angstrom resolution along the c-axis, the re flections were weak, For accurate measurement of these reflection inte nsities, 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 o n a data set from a PtCl4 derivative.