6 research outputs found
Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells
AbstractA 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 a = b = 105.6 Å, c = 506.9 Å. 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 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 Å along the c∗-axis, whereas along the a∗–b∗-plane reflections were limited to 6.6 Å. Data analysis is under way on a data set from a PtCl4 derivative
Reconstitution of Rabbit Skeletal Muscle Troponin from the Recombinant Subunits All Expressed in and Purified from E. coli
Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are β-TnT(N'–208), TnI(C64A, C133S), and the wild type TnC. β–TnT(N'–208) is a 25 kDa fragment of y9-troponin T, which consists of 208 amino acids and lacks 58 residues in the N–terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys–64 and Cys–133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-Sl ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials
Lobster troponin C: Amino acid sequences of three isoforms
We have determined the amino acid sequences of the three major isoforms of lobster troponin C, using protein chemistry methods. Each isoform contains 150 amino acid residues, with a calculated Mr of 17,000 ± 100. Sequence differences occur in all regions of the polypeptides, indicating that the isoforms are products of three different genes, rather than alternative transcripts of a single gene. Analysis of the sequences predicts that functional Ca2+-binding sites are present only in regions II and IV of four ancestral regions