Molecular interactions during the assembly of cowpea chlorotic mottle virus studied by magnetic resonance

G. Vriend

Research output: Thesisinternal PhD, WU

Abstract

<p/>This thesis describes the application of <sup>1</sup> H- and <sup>13</sup> C- NMR, EPR, ST-EPR and calculational methods to study cowpea chlorotic mottle virus. This virus consists of RNA encapsidated by 180 identical protein subunits, arranged icosahedrally. The isolated coat protein of cowpea chlorotic mottle virus can be brougth into several well defined states of aggregation. This study could be carried out, because these stages can be produced in quantities sufficient to allow magnetic resonance measurements. All the results obtained are combined in the following model for the assembly proces of the virus:<p/><img src="/wda/abstracts/i956_1.gif" height="190" width="600"/><p/>In this model a rigid protein core with a highly mobile, basic, N- terminal arm is invoked. This arm is the RNA binding part of the protein. The high mobility of this arm enhances the probability of interaction with the RNA and enables the protein to exhibit different modes of bonding to different local structures of the RNA. Upon binding the RNA, the N-terminal arm adopts a rigid a-helical conformation.<p/>In chapter two it is shown that virtually no mobility on a timescale faster than 10 <sup>-7</sup> s can be observed in the virus.<br/>In chapter three an EPR and ST-EPR study is presented on spin-labelled virus, From the results it is concluded that no anisotropic subunit mobility is present in the virus on the 10 <sup>-5</sup> -10 <sup>-7</sup> s timescale. Also in this chapter it is suggested that many ST-EPR results on maleimide- labelled proteins, in which anisotropic protein mobility was invoked for the interpretation of the spectra, are based on artefacts.<br/>Chapter four shows that the N-terminal arm is the RNA binding part of the protein. This arm is found to be very mobile in the absence of RNA, whereas immobilization occurs upon binding RNA.<br/>Chapter five is a small excursion to other plant viruses. It is shown that brome mosaic virus and belladonna mottle virus show the same behaviour as cowpea chlorotic mottle virus, whereas cowpea mosaic virus behaves completely different.<br/>Chapter six deals with the molecular interactions during the assembly process. In this chapter it is shown that the arginine and lysine containing part of the N-terminal arm is responsible for the binding of oligo-nucleotides. It is suggested that a double stranded nucleotide conformation is required for proper interaction.<br/>In chapter 7 a secondary structure prediction of the coat protein of CCMV is presented. The results indicate that the N-terminal arm shows no structural preference when the positive charges of arginine and lysine are retained, whereas in the absence of these positive charges there is a tendency towards α-helix formation. Also it is concluded that cowpea chlorotic mottle virus possesses the same β-role topology as southern bean mosaic virus, sattelite tobacco necrosis virus and tomato bushy stunt virus.<br/>Chapter eight presents the results of an energy calculation study on the N-terminal arm. Although the method used contains too many approximations to allow detailed conclusions, the results of chapter seven are fully confirmed by these calculations.<br/>In the model for the protein-RNA interaction in CCMV <em></em> which is presented in chapter five, a random coil conformation for the N-terminal protein arm is invoked. In chapter nine an extension of this part of the model is presented. It is shown that the N-terminal possesses some time-averaged secondary structure (presumably an α-helix) in the absence of RNA. A conformational exchange process is observed in the N-terminal arm, in which about half 'of the residues participate. In empty capsids not all the N-terminal arms turn out to be mobile. 20-55% of the N-terminal arms is immobile on the NMR timescale in the empty capsids of the coat protein of CCMV.<p/>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Schaafsma, T.J., Promotor
  • Heeminga, M.A., Co-promotor, External person
Award date14 Oct 1983
Place of PublicationWageningen
Publisher
Publication statusPublished - 1983

Keywords

  • cells
  • cowpeas
  • plant viruses
  • vigna
  • viruses

Fingerprint Dive into the research topics of 'Molecular interactions during the assembly of cowpea chlorotic mottle virus studied by magnetic resonance'. Together they form a unique fingerprint.

  • Cite this