Dimerization of recombinant tobacco mosaic virus movement protein - PubMed (original) (raw)

Dimerization of recombinant tobacco mosaic virus movement protein

Laurence M Brill et al. J Virol. 2004 Apr.

Abstract

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.

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Figures

FIG. 1.

FIG. 1.

Preparative SDS-PAGE was used to isolate soluble MP-FL. (A) SDS-PAGE of every fifth fraction. (B) MP-FLP1 contained isolated MP monomer, in contrast with chromatographic peaks P1 and P2 (6), which contained MP aggregates, monomer, and fragments. Mr, molecular mass standards in kilodaltons (A and B). (C) Migration of MP-FLP1 and MP-FLP2 in native PAGE suggested that the MP was essentially monodisperse. Numbers above the first five lanes denote the molecular masses of protein standards (in kilodaltons): 14, α-lactoglobulin; 29, carbonic anhydrase; 45, egg white albumin; 66 and 132, BSA monomers and dimers (and some higher-order oligomers); 272 and 545, urease trimers and hexamers. Lanes containing MP-FLP1 or MP-FLP2 are indicated. Asterisks mark urease trimer and hexamer bands. An arrowhead points to the MP. Note that migration was not necessarily proportional to the molecular mass in native PAGE (panel C only). Gels were stained with Coomassie brilliant blue R-250.

FIG. 2.

FIG. 2.

Equilibrium analytical ultracentrifugation of MP-FLP1 and MP-FLP2. Residuals (upper panels) and _A_260 (lower panels) versus radius are shown. Molecular mass estimates are also shown for MP-FLP1 (A) and MP-FLP2 (B). The data are fitted very well by a one-state model, suggesting that the 30-kDa MP-FL is monodisperse and dimeric in TN buffer containing 0.05% βOG.

FIG. 3.

FIG. 3.

Isolated core domain of MP from P1 (MP-CP1) was composed of monomers and dimers. Migration of preparations of MP-FLP1 and MP-CP1 in analytical SDS-PAGE (A) and native PAGE (B). Protein standards and asterisks are as described in the legend to Fig. 1. (C) Equilibrium analytical ultracentrifugation of MP-CP1. The results are fitted very well by a two-species model, suggesting that the MP core exists primarily as monomers and some dimers.

FIG. 4.

FIG. 4.

CD spectroscopy suggested that the α-helical content of MP-CP1 was slightly lower than that of MP-FLP1 and MP-FLP2. MP was at 1 mg/ml in TN buffer containing 0.05% βOG. Molar ellipticity is shown from 200 to 250 nm; shorter-wavelength data were unreliable due to UV absorption by the buffer.

FIG. 5.

FIG. 5.

Topological model of the MP dimer refined from that of Brill et al. (6). Hydrophobic amino acid residues are yellow, basic residues are blue, acidic residues are red, and Cys residues are green. The trypsin-resistant core contains the first 249 or 250 amino acid residues, including the peptide (gray bar) that was used to produce anti-MP antibodies used previously. The acidic C terminus (residues 249 to 268) was rapidly removed by trypsin (6) to yield the trypsin-resistant core domain (Fig. 3 and 4). As defined by Saito et al. (34), domain C (residues 252 to 268) is acidic (red bar) and domain B (residues 206 to 250) is basic (blue bar). Cytoplasmic, transmembrane, ER lumenal, and core domains were previously proposed (6). Transmembrane domains are presumed to be α-helical. Ser37 (19), Ser258, Thr261, and Ser265 (39) were reported to be phosphorylation sites (pink circles). Because dimerization (Fig. 3C) and α-helicity (Fig. 4) decrease as the full-length protein is converted to the core lacking the C terminus, we propose that the acidic C terminus participates in dimerization. Dimerization could be mediated in part by charge-charge interactions between acidic region C and basic region B of another monomer.

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