Structural studies on alfalfa mosaic virus coat protein
Abstract
Fukuyama et al., 1983 (J. Mol. Biol. 167 873-894) reported the structure of a T = 1 icosahedral particle assembled from the capsid protein of Alfalfa Mosaic Virus. The information contained in the 4.5 A resolution structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral 5-fold axes and a proposal that the capsid protein adopts a $\beta$-barrel structure. The subunit fold or the main chain trace was not definitively determined. In the present work, the x-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by Hata, Fita & Rossmann (unpublished) were reprocessed to 4.0 A resolution and the structure solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the $\beta$-sandwich fold and provided information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4 A resolution. To extend the resolution of the current structure, crystals have been grown from the T = 1 particles assembled from the coat protein expressed in E. coli. A truncated version of the coat protein, in which the first 25 residues have been genetically removed, has also been expressed and again, crystals grown from the assembled T = 1 particles. These crystals did not diffract x-rays better than the existing data of Fukuyama et al. Efforts are continuing to refine crystallization conditions to obtain crystals that would diffract x-rays to higher resolution. The subunit polyalanine structure was used to construct a model for the bacilliform AMV particle which adopts a P6 cylindrical structure capped by icosahedral halves. The bacilliform AMV particles as well as the coat protein-DNA complexes are being studied by electron microscopy and image reconstruction methods. Protocols to perform model assisted 3-D reconstructions of non-icosahedral, non-helical particles with EM images have been developed and are being used in the study of AMV particles. The structure of T = 1 icosahedral AMV particle has also been determined by cryo electron microscopy and image reconstruction methods and found to be in excellent agreement with the x-ray model.
Degree
Ph.D.
Advisors
Johnson, Purdue University.
Subject Area
Biophysics|Biochemistry|Molecular biology
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