Conformation of the RNA-binding N-terminus of the coat protein of cowpea chlorotic mottle virus : a nuclear magnetic resonance and optical spectroscopy study

M. van der Graaf

Research output: Thesisinternal PhD, WU

Abstract

<p>The objective of the study described in this thesis was to obtain information about protein-RNA interactions in cowpea chlorotic mottle virus (CCMV). CCMV consists of RNA and a protective protein coat, composed of 180 identical coat proteins. The positively charged N-terminal arm of the coat protein is essential for binding the encapsidated RNA. Previously, the so-called 'snatch-pull' model has been suggested for the assembly of coat protein and RNA. According to this model the N-terminal region has a flexible random-coil conformation in the absence of RNA, but attains an a-helical conformation upon RNA-binding. In the present study a synthetic peptide containing the first 25 amino acids of CCMV coat protein (P25) was used as a model for the N- terminus, and oligophosphates and oligonucleoticles were used as models for the viral RNA. The conformation of the peptide was studied by nuclear magnetic resonance (NMR) and circular dichroism (CID). Changes in the conformation of the oligonucleotides were studied by CD and absorbance spectroscopy. The results confirm the snatch-pull model and allow an extension to a more detailed model for the assembly of CCMV coat protein and RNA (see below).<p>Chapter 2 describes the effects of ionic strength, addition of (oligo) phosphates, and temperature on the conformation of the peptide P25 containing six arginines and three lysines. CD experiments show that the peptide has 15-18% α-helical and about 80% random coil conformation in the absence of inorganic salt at 25°C. Lowering the temperature to 10°C increases the a-helix content to 20-21%. Addition of inorganic salts results in a larger increase of the amount of α-helical conformation, up to 42% in the presence of oligophosphate with an average chain length of eighteen phosphates. One-dimensional proton NMR experiments show that the α-helix formation starts in the region between Thr9 and GIn12, and is extended in the direction of the C-terminus. NMR relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 at the beginning of the α-helical region.<p>Chapter 3 gives a description of two-dimensional proton NMR experiments on P25 in the presence of sodium monophosphate. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly interconverting to more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium towards the helical structures. At 10°C a perceptible fraction of the conformational ensemble consists of structures with an a-helical conformation between residues 9 and 17, likely starting with a turn- like structure around Thr9 and Arg10. The region close to Arg 10 shows the strongest tendency to attain an α-helical conformation.<p>Chapter 4 presents a two-dimensional proton NMR study on the effect of (oligo)phosphates on the conformation of P25. NMR experiments were performed on the highly positively charged peptide in the presence of an excess of monophosphate, tetraphosphate or octadecaphosphate. The peptide alternates between various extended and helical structures in the presence of monophosphate, but this equilibrium shifts towards the helical structures in the presence of oligophosphates. In the presence of tetraphosphate the α-helical region is situated between residues 10 and 20. NMR distance constraints obtained for P25 in the presence of tetraphosphate have been used to generate peptide structures by distance geometry calculations. These calculations resulted in eight structures belonging to two structure families. The first family consists of five structures with an α-helix-like conformation in the middle of the peptide, and the second family consists of three structures with a more open conformation. The presence of two structure families indicates that even in the presence of tetraphosphate the peptide is flexible.<p>Chapter 5 describes a two-dimensional proton NMR study on the intact coat protein of cowpea chlorotic mottle virus (molecular mass: 20.2 kDa) present as dimer (pH = 7.5) or as capsid consisting of 180 protein monomers (pH = 5.0). The spectra of both dimers and capsids show resonances originating from the flexible N-terminal region of the protein. The complete resonance assignment of P25 made it possible to interpret the spectra in detail. The capsid spectrum shows backbone amide proton resonances arising from the first eight residues having a flexible random coil conformation, and side- chain resonances arising from the first 25 N-terminal amino acids. The dimer spectrum shows also side-chain resonances of residues 26 to 33, which are flexible in the dimer but immobilized in the capsid. It is suggested that the carboxyl group of GIu33 plays a role in the pH-dependent association of the coat protein. Neutralization of this acidic residue by low pH or by the presence of positively charged bases of the RNA possibly results in a conformation necessary for capsid formation. The NMR experiments indicate that the conformation of the first 25 amino acids of the intact viral coat protein in dimers and capsids is comparable to the conformation of the synthetic peptide P25.<p>Finally, Chapter 6 deals with the effect of P25 on the structure of oligonucleoticles. The experiments described in this chapter have been carried out on samples containing micromolar concentrations of both P25 and oligonucleoticles. For NMR measurements samples with concentrations of more than 100 times higher are necessary. Unfortunately, it was not possible to avoid precipitation at such high concentrations of P25 and oligonucleotides in aqueous solution. However, conformational changes of the oligonuclecitides r(A) <sub>12</sub> , d(GC) <sub><BLINK>5</BLINK></sub> , and d(AT) <sub>5</sub> upon binding to P25 could be studied by using absorption and CD spectroscopy. r(A) <sub>12</sub> has a single-stranded structure at pH = 7.2, and exists as a protonated duplex at pH-values below 5.8. In this duplex a phosphate oxygen of the RNA backbone forms a hydrogen bond to the amino group of the adenine. The oligonucleoticles d(GC) <sub>5</sub> and d(AT) <sub>12</sub> form duplexes with Watson-Crick base-pairing. The results show that P25 has only a minor effect on the single-stranded structure of r(A) <sub>12</sub> at pH 7.2, but disrupts the double-stranded structure of r(A) <sub>12</sub> at pH = 5.0. At pH = 4.0, the double stranded structure of r(A) <sub>12</sub> is stabilized by protonation of the adenine at the N <sub>1</sub> position and the peptide is not able to disrupt the double-stranded structure. The double-stranded structures of d(GC) <sub>5</sub> and d(AT) <sub>5</sub> with Watson-Crick base-pairing are stabilized upon binding to the peptide. All conformational changes observed indicate that the positively charged side chains of the peptide have strong electrostatic interactions with the negatively charged phosphate groups of the oligonucleoticles.<p>The results of the study described in this thesis confirm the snatch-pull model mentioned in the beginning of this summary. Although P25 alternates between various conformations in aqueous solution, the region between residues 10 and 20 shows a tendency to adopt an α-helical conformation. This α-helical structure is stabilized by low temperature, high ionic strength, and the addition of (oligo)phosphates, mimicking the RNA. It has been shown that the α-helix formation starts in the region between Thr9 and GIn12. Based upon this observation, a more detailed model for the assembly of CCMV coat protein and the viral RNA is proposed. It is suggested that hydrogen bond formation by the side chains of Thr9 and GIn12 initiates α-helix formation. After this initiation, the helix is extended in the direction of the C-terminus upon binding of negatively charged phosphate groups to the positively charged side chains present in the N-terminal region of the coat protein. The results indicate a strong effect of the presence of phosphates on Arg10. Phosphate binding to this residue may extend the helix in the direction of the C-terminus because of the removal of an unfavourable interaction between the positive charge at this position and the macrodipole of the helix. The extension of the helical region results in a proper orientation of the positively charged side chains for binding to the phosphate groups of the RNA backbone. In an alfa- helical conformation the distance between the arginines at positions 10, 14, 18 and 22 (- 6 Å) is comparable to the distance between two neighbouring phosphate groups in an A-type RNA helix (- 5.9 Å).
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Schaafsma, T.J., Promotor
  • Hemminga, M.A., Promotor, External person
Award date2 Jun 1992
Place of PublicationS.l.
Publisher
Publication statusPublished - 1992

Keywords

  • cowpea mosaic virus
  • virology
  • molecular biology
  • physical chemistry
  • physics
  • proteins
  • spectroscopy
  • spectral analysis
  • analytical methods
  • rna
  • nuclear magnetic resonance
  • nuclear magnetic resonance spectroscopy
  • optics

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