Structural characterization of Tobacco etch virus coat protein mutants.
The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli. To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.
Voloudakis
A E
AE
Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
Malpica
C A
CA
Aleman-Verdaguer
M-E
ME
Stark
D M
DM
Fauquet
C M
CM
Beachy
R N
RN
D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D017354:Point Mutation; D017800:Potyvirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D014026:Nicotiana / Q000821:virology
D036022:Capsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins