Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein - PubMed (original) (raw)
Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein
L M Brill et al. Proc Natl Acad Sci U S A. 2000.
Abstract
The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.
Figures
Figure 1
SDS/PAGE of inclusion bodies from E. coli pET3MP. Labeling below the gels indicates the concentration of urea and the sample identity as unfractionated (u), supernatant (sup; s), or pellet (pel; p). * indicate a high-speed pellet or high-speed supernatant. Lanes 1 and 2 contain whole-cell lysates before and after MP gene induction in E. coli pET3MP, respectively. Lanes 3–8, inclusion bodies isolated by using a previous protocol (ref. ; labeled older). Lane 3, soluble in buffer L with 4 M urea (25). Lane 4, insoluble in buffer L with 4 M urea but soluble in buffer L with 8 M urea. Lane 5, material from lane 3 in buffer L without urea (25). Lanes 6–8, sample from lane 5 fractionated via centrifugation; lane 6, low-speed (15,000 g) pellet; lane 7, high-speed (100,000 g) pellet; lane 8, high-speed supernatant. Lane 9, inclusion bodies isolated by using the protocol described in this paper (labeled new). SDS/PAGE gels were stained with Coomassie. Molecular mass standards (_M_r) in kDa. Lanes 1 and 2 were from one gel, and lanes 3–9 were from another gel. The order of lanes 3 and 4 was changed for clarity.
Figure 2
Purification and characterization of recombinant MP. (A) Anion exchange chromatography. Conductivity, 13.2–71.4 mS; pH, 8.73–9.24. P1, P2, and P3, peaks 1, 2, and 3. (B) Chromatographic fractions 1–40. (Upper) Silver-stained SDS/PAGE gels; (Lower) Western blots detecting MP. Unheated samples were subjected to SDS/PAGE; _M_r as in Fig. 1. (C) Absorption spectra of P1, P2, and P3. (D) Nuclease digestion. Ethidium bromide-stained 1% Tris/borate/EDTA (lanes 1–4) and 1.5% formaldehyde-agarose (lanes 5–12) gels. Lanes 1–4: pET3MP DNA; in DNaseI buffer only (lane 1), or RNaseA buffer only (lane 2); in buffer with DNaseI (lane 3), or RNaseA (lane 4). Lanes 5–8: TMV RNA; in DNaseI buffer only (lane 5), or RNaseA buffer only (lane 6); with DNaseI (lane 7), or RNaseA (lane 8). Lanes 9–12: concentrated P3; in DNaseI buffer only (lane 9), or RNaseA buffer only (lane 10); with DNaseI (lane 11), or RNaseA (lane 12). Standards, in kbp (DNA; Left) and kb (RNA; Right).
Figure 3
CD spectroscopy of solubilized MP in the presence of urea and SDS indicated high α-helical content. The MP concentration was 17 μM, in TNEM2MU + 0.1% SDS. MRE, mean residue ellipticity.
Figure 4
SDS/PAGE (A) and Western blots (B) of MP incubated with trypsin showed that a core domain resists cleavage. Lanes 1, MP inclusion bodies (MPIB) in TNEM8MU buffer. Lanes 2, P1, and lanes 3, P2 (see Fig. 2_A_). Lanes 4–14, MP from P2 in TNETMG buffer. Lanes 4, control incubation, no trypsin (NT). Lanes 5–14 were incubated with trypsin for the indicated times, in min. _M_r as in Fig. 1; m, monomer; c, core. Ponceau-S staining showed even transfer of proteins to membranes. Trypsin (<2 ng per lane) was not detected in the gels.
Figure 5
MS of the full-length MP monomer (A) and core domain (B; Fig. 4). Molecular masses of peptides (short lines) generated by extensive trypsin digestion matched the predicted masses deduced from the MP sequence. Two hydrophobic peptides containing putative transmembrane domains (residues 39 or 58–85, and 145–175; open bars) were rarely detected. The core (B) was generated by release of residues 250–268 from the C terminus (open bar) by cleavage at highly sensitive K residues (*). Other K and R residues were more resistant to cleavage (arrowheads) and some were not cleaved (arrows). In some experiments, residues 1–6 of the monomer (A) were detected. The profiles are representative of MP from P1 and were similar for P2.
Figure 6
Topological model of the TMV MP. The amino acid sequence was deduced from the ORF encoding the MP (32). Hydrophobic amino acid residues are yellow, basic residues are blue, acidic residues are red, and Cys residues are green. The trypsin-resistant core domain contains the first 249 or 250 residues, including the peptide (boxed) that was used to produce MP antibodies used in Figs. 2_B_ and 5_B_. The C terminus (gray) was rapidly removed by trypsin. As previously defined (36), domains I (residues 56–96) and II (residues 125–164) are regions that are conserved among tobamovirus MPs and are outlined in black; domains A (residues 183–200) and C (residues 252–268) are acidic and are outlined in red; and domain B (residues 206–250) is basic and is outlined in blue. Cytoplasmic, transmembrane, ER luminal, and core domains were inferred from Western blots of proteinase K-treated microsomes, hydropathy analysis, fluorescence microscopy (16, 20, 22, 24), trypsin susceptibility, and MS. Transmembrane domains are presumed to be α-helical. Hydrophobic peptides (39–85 vs. 58–85) differed in length between the monomer and the core (Fig. 5), suggesting that the C terminus of the monomer may interact with its N terminus. Serine-37 is phosphorylated (P3-; ref. 37), and S 218 may be phosphorylated (P3-?) based on Prosite prediction and sequence conservation among tobamovirus MPs (not shown). Met residues (*) have been reported to generally be involved in protein–protein interactions (38).
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