Studies of Virus Structure by Laser-Raman Spectroscopy
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Studies of Virus Structure by Laser-Raman Spectroscopy. 3. Turnip Yellow Mosaic Virus T. A. Turano, K. A. Hartman,* Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, Rhode Island 02881
and G. J. Thomas, Jrn2 Department of Chemistry, Southeastern Massachusetts University, North Dartmouth, Massachusetts 02747 (Received December 29, 1975)
Laser-excited Raman spectra of a plant virus, the turnip yellow mosaic virus (TYMV), reveal vibrational frequencies characteristic of both the nucleic acid (RNA) and coat-protein components. The prominent Raman lines of the virus were assigned to specific subgroups of the constituent macromolecules and were examined as a function of temperature for both HzO and DzO solutions. The Raman data indicate the following structural features of aqueous TYMV. Amide I and I11 frequencies of the coat-protein molecules exclude the presence of appreciable amounts of both a-helical and antiparallel-,&sheet structures and suggest that the polypeptide chain is mostly in an irregular conformation. All of the four cysteine residues of the coat-protein molecule have S H groups and no S-S linkages exist in TYMV. All SH groups are accessible to the solvent, as evidenced by their deuterium exchange. A tentative finding is that many or all of the carboxyl groups of aspartic and glutamic acid residues in the coat protein are ionized a t pH 7. Tryptophan residues are exposed to sdlvent H2O molecules, whereas tyrosine residues are apparently not in contact with the solvent and form strong hydrogen bonds between the tyrosyl -OH donor and negative acceptor groups within the virion. These structural properties of TYMV are unchanged over the temperature range 0-54 “C, above which the virus structure collapses. The encapsulated RNA molecule of TYMV contains an unusually low amount of ordered secondary structure (-60%), in comparison with those other single-stranded RNA species which have been studied (-85%). The secondary structure of encapsulated RNA is also largely resistant to changes in temperature up to 54 “C. The cytosine residues of TYMV RNA are not protonated at pH 7, either for protein-free RNA or for RNA encapsulated within the virus. It is therefore unlikely that specific hydrogen bonding interaction between cytosine residues of RNA and carboxyl groups of coat proteins are a major source of stabilization of the native TYMV virion, a t pH 7.
Introduction Considerable progress has been made recently in the application of laser-Raman spectroscopy to the elucidation of the structures of biological molecule^.^^^ Raman spectra of the constituents of nucleic acids were first observed in the laboratory of Professor R. C. Lord in the early 1960’s (for a discussion of this work see ref 3-5). More detailed investigations of polynucleotides and naturally occurring nucleic acids soon followed6-9 as more powerful laser sources became generally available. Lord and Yule next began the modern era of laser-Raman spectroscopy of proteins, by assigning spectral lines of the amino acids and successfully utilizing their results in an analysis of the enzyme, lysozyme. In addition to the study of other proteins, Chen and Lordll in 1974 produced a correlation between the chain conformations of model polypeptides and their corresponding Raman lines. This work suggested the use of conformationally sensitive Raman frequencies of the peptide group, the so-called amide frequencies, as a means of estimating protein conformation without necessitating a detailed and laborious crystallographic analysis. In 1973 enough data had been accumulated to allow Hartman, Clayton, and Thomas12 to initiate the use of laserRaman spectroscopy in nucleoprotein research by obtaining spectra of the bacterial virus, R17, and assigning its individual Raman lines to vibrations of specific subgroups of viral RNA and coat protein. In 1975, Thomas and Murphyl3 and Thomas, Prescott, Ordzie, and Hartman14 studied the DNA viruses, Pfl and fd, and the RNA virus, MS2, respectively.
Several structural properties of the nucleic acid and protein components of these viruses were revealed by the Raman data. The advantages of laser-Raman spectroscopy in investigations of viruses and other nucleoproteins are that small amounts of sample are required, that a wealth of information is available from the many vibrational scattering lines assignable to subgroups of both nucleic acid and protein components, and that the entire vibrational spectrum is open to analysis for aqueous (HzO and D20) solutions of viruses. Structural information of the kind obtained from Raman spectra of viruses is difficult or impossible to obtain by other means. It is for these reasons that we have undertaken structural studies of viruses, including the turnip yellow mosaic virus (TYMV), using laser-Raman spectroscopy. A single TYMV particle (virion) contains about one-third by weight nucleic acid (one molecule of RNA of molecular weight, mol wt = 1.91 X IO6) and about two-thirds by weight protein (180 molecules of “coat” protein, each of mol wt = 20 133, which together form the capsid).15 TYMV has been extensively studied by x-ray crystallography, electron microscopy, and various chemical and hydrodynamic methods.15 These analyses reveal the gross morphological properties of the capsid, but indicate virtually nothing about the locus or conformation of the encapsulated RNA. The finer details of the capsid structure are also unknown. For example, the x-ray diffraction data are accounted for by an icosahedral capsid assembled from 20 hexamers and 12 pentamers of the coat-protein monomer. However, the conformations of the coat-protein molecules and the nature The Journal of Physical Chemistry, Vol. 80,No. 11, 1976
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of the interactions between them in the assembled capsid are questions still unanswered. Evidence suggests that the RNA of the virion laces in and out between the coat-protein molecules. In a model proposed by Kaper15 the RNA is considered to be bound to the capsid protein by hydrogen-bonding interaction between protonated cytosine residues of the RNA and protonated carboxyl groups (of aspartic and/or glutamic acid residues) of the protein (pH
