Recombinant tobacco mosaic virus movement protein is an RNA-binding, α-helical membrane protein

Recombinant tobacco mosaic virus movement protein is an RNA-binding, α-helical membrane protein

^*Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, Mail Drop MB 28, La Jolla, CA 92037; and ^‡Division of Cardiovascular Diseases, Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Roger N. Beachy

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 ≈70% α-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 α-helical transmembrane domains and a protease-sensitive carboxyl terminus.

Footnotes

↵ † Present address: Pioneer Hi-Bred International, Inc., 7250 N.W. 62nd Avenue, P.O. Box 552, Johnston, IA 50131-0552.
↵ § To whom reprint requests should be sent at present address: Donald Danforth Plant Sciences Center, 7425 Forsythe Boulevard, Box 1098, St. Louis, MO 63105. E-mail: rnbeachy{at}danforthcenter.org.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.130187897.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.130187897
Abbreviations:

TMV,

tobacco mosaic virus;

MP,

movement protein;

TNETMG buffer,

50 mM Tris, pH 9.0/700 mM NaCl/1 mM EDTA/1% (vol/vol) Tween 20/10 mM 2-mercaptoethanol/10% glycerol;

TNEM8MU buffer,

10 mM Tris, pH 9.0/500 mM NaCl/5 mM EDTA/1 mM 2-mercaptoethanol/8 M urea;

TNEM2MU buffer,

identical to TNEM8MU buffer except contains 2 M urea;

ss,

single-stranded;

ER,

endoplasmic reticulum;

K,

lysine;

R,

arginine