Computational protocol: Three mammalian tropomyosin isoforms have different regulatory effects on nonmuscle myosin-2B and filamentous β-actin in vitro

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[…] To generate the expression construct for the heavy chain of the double-headed NM-2B–HMM construct with C-terminal octahistidine and Avi tags, the DNA sequence that encodes amino acids 1–1344 was PCR-amplified using human cDNA as a template. The primers used were 5′-CGCGCGGCGCGCATGGCGCAGAGAACTGGACTCGA-3′ and 5′-CAGCTGGTCGACGTGGTGATGATGATGATGATGATGCTCCTCTTCCAGCTGCCGGAT-3′ containing restriction sites for BssHI and SalI, respectively. The pFast BacTM vector containing the NM-2B heavy chain sequence and the pFastBacTM Dual vector containing the sequences for MYL6 and MYL12b were cotransformed for protein production in SF9 cells. Vectors and cells were obtained from Thermo Fisher Scientific. The generation of the expression vector encoding the motor domain of human NM-2B0 (residues 1–782), two Dictyostelium discoideum α-actinin repeats, and a C-terminal octahistidine tag was described elsewhere (). Sf9 cells were transfected with the recombinant bacmids using the FuGENE HD transfection reagent (Promega). To purify the free MYL6 and MYL12b light chains, the genes were cloned into the pGEX expression vector (GE Healthcare Life Sciences) for protein production in E. coli.Expression constructs for rat Tpm1.8cy (b.–.b.d) (NCBI reference ID NP_001029245.1), rat Tpm1.12br (b.–.b.c) (NCBI reference ID NP_001288665.1), and human Tpm3.1cy (b.–.a.d) (NCBI reference ID NP_705935.1) were cloned into pET23a+ for expression in E. coli strain RosettaTM (Merck). We refer to the different tropomyosin isoforms in the text mostly by their short name in accordance with a recently introduced systematic nomenclature (). Full formal protein names are used when we refer to differences in exon usage between the isoforms. Alternate names and changes in exon usage are listed in . Moreover, we refer to the unassembled double-stranded coiled-coil protein as dimer. Cytoskeletal Tpm isoforms form predominantly homopolymers of Tpm homodimers. The extensions “br” and “cy” in the formal protein name indicate prominent but not exclusive associations with brain tissue and cytoskeletal structures. Tpm1 or Tpm3 specifies the protein is encoded by TPM1 or TPM3. The four-letter code extension of the formal protein name indicates splice form usage for the variable exons 1, 2, 6, and 9. All three isoforms lack the region encoded by exon 2 (). The short names Tpm1.8, Tpm1.12, and Tpm3.1 are used throughout the text, unless we refer to the splicing of the four exons that vary in vertebrates.Purification of chicken skeletal muscle α-actin () and recombinant human β-actin was performed as described previously (). Pellets of Sf9 cells producing NM-2B0 motor domain or HMM constructs were suspended in lysis buffer (50 mm HEPES, pH 7.5, 200 mm NaCl, 10 mm β-mercaptethanol, 4 mm MgCl2, 4 mm ATP, 1 mm EGTA, 0.5% Triton X-100, and cOmpleteTM protease inhibitor mixture (Merck). The cell suspension was sonicated four times for 1 min at 40% power and 50% duty cycle (Bandelin Sonoplus, Berlin, Germany). The lysate was centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was loaded onto a Ni2+–nitrilotriacetic acid affinity column (Qiagen) equilibrated with lysis buffer. The column material was washed with 10 column volumes lysis buffer and 10 column volumes wash buffer (25 mm HEPES, pH 7.3, 200 mm NaCl, 0.5 mm EGTA, 3 mm MgCl2, 65 mm imidazole). The protein was eluted with 6 column volumes elution buffer (25 mm HEPES, pH 7.3, 200 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm DTT, and 200 mm imidazole). In addition, the free forms of human recombinant ELC (essential myosin light chain) (MYL6) and RLC (regulatory myosin light chain) (MYL12b) constructs were produced in E. coli. The purified light chains were added at 1 μm concentration to the elution buffer used for the purification of the HMM construct. Individual fractions were analyzed by SDS-PAGE, concentrated, and dialyzed overnight against dialysis buffer (25 mm HEPES, pH 7.3, 200 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm DTT, and 3% trehalose). Dialyzed protein was applied to a HiLoad Superdex 200 prep grade 16/60 gel filtration column (GE Healthcare Life Sciences) equilibrated with dialysis buffer. The resulting protein fractions were concentrated to a final concentration of ∼6 mg ml−1. The purified protein was supplemented with 7% (w/v) trehalose, flash frozen in liquid nitrogen, and stored at −80 °C.Tpm production was induced by the addition of 1 mm isopropyl β-d-thiogalactopyranoside, followed by incubation with vigorous shaking for 3–4 h at 37 °C. In addition to human Tpm3.1, we produced rat isoforms Tpm1.8 and Tpm1.12. Changes between rat and human Tpm1.8 and Tpm1.12 occur at S30T, Q37H, and K184R. In the case of Tpm1.12, a fourth change corresponds to H226Q. All changes occur in the c position of the repeating heptad repeat pattern (). Residues in the c position are located at the outside of the coiled-coil and contribute to stability through α-helical propensity (). The program Paircoil2 () predicts the parallel coiled-coil fold of Tpm1.8 and Tpm1.12 to be unchanged by these species specific substitutions. The recombinant, tag-free Tpm constructs were purified by ion exchange chromatography as described by Coulton et al. () with minor modifications. Purified Tpm was stored in 5 mm potassium phosphate, pH 7.0, 100 mm NaCl, and 5 mm MgCl2. Protein concentration was determined using the Bradford assay and protein absorbance readings at 276 nm. Measurements were performed with a UV-2600 spectrophotometer (Shimadzu, Duisburg, Germany). Recombinant myosin light chain kinase was obtained from Sigma–Aldrich. […]

Pipeline specifications

Software tools MUSCLE, Paircoil
Applications Protein structure analysis, Nucleotide sequence alignment
Organisms Homo sapiens
Chemicals Adenosine Diphosphate, Phosphates