Biomedical Engineering Reference
In-Depth Information
due to the recognized diculties of characterizing intact viruses, particularly
the encapsidated nucleic acid, using X-ray diffraction and NMR. As an il-
lustration of one strategy for characterizing both the protein capsid and the
encapsidated nucleic acid of a virus using ROA, we present in Fig. 7.9 results
for cowpea mosaic virus (CPMV) [60].
CPMV is the type member of the comovirus group of plant viruses and is
a bipartite virus with the genome consisting of two different RNA molecules
(RNA-1 and RNA-2) separately encapsidated in identical icosahedral cap-
sids. The structure of the protein capsid is known to be constructed from 60
copies of an asymmetric unit made up of three different protein domains each
of which supports a 'jelly-roll
-sandwich' fold [64-66]. However, the struc-
tures of RNA-1 and RNA-2 were not resolved. Preparations of CPMV can be
separated into empty protein capsids, capsids containing RNA-1 and capsids
containing RNA-2. The top panel of Fig. 7.9 shows the Raman and ROA
spectra of the empty protein capsid, with the spectral band patterns being
characteristic of the jelly-roll
β
-sandwich fold of the capsid proteins. The mid-
dle panel shows the spectra of the capsid containing RNA-2, with bands from
the nucleic acid now evident as well as those from the protein. Though not
shown here, the Raman and ROA spectra for the capsid with RNA-1 were
very similar. The bottom panel shows the spectra obtained by subtracting
the top from the middle spectra, leading to Raman and ROA spectra for the
encapsidated RNA-2 plus any changes in protein bands due to protein-RNA
interactions [59]. The difference ROA spectrum is very similar to those of syn-
thetic and natural RNA molecules and therefore probably originates mainly
in the viral RNA: the details reflect the single-stranded A-type helical con-
formation of the encapsidated RNA-2, which was not previously known. The
ROA spectrum of RNA-1 was extracted using the same procedure and was
found to be very similar to that of RNA-2, despite having a quite different
length and base sequence. This suggests that both RNAs take up the same
'default' conformation within the capsid, possibly because the protein-nucleic
acid interactions in this particular virus are very weak.
β
7.9 Developments in Computational Spectral Analysis
It is likely that many of the most exciting advances in ROA over the next few
years will be in computational approaches. In particular, the implementation
of basis functions and computational procedures for modelling ROA spectra
in both the GAUSSIAN and DALTON suites has recently provided several
new insights into ROA spectra-structure relationships [67-71]. However, we
will not review such computational modelling work here but summarize the
application of chemometric tools for analysing measured ROA spectra.
Search WWH ::
Custom Search