Biology Reference
In-Depth Information
..1 Solvent-exposed lys Side Chains on CPMV
Nearly all VNPs currently in use for nanotechnology offer solvent-
exposed addressable Lys side chains. Functionalization and attachment is
straightforward, and many reagents for Lys-modification are commercially
available. For an overview of VNPs with addressable surface Lys residues,
the reader is referred to Table 4.1 at the end of this chapter.
Bioconjugation
has
been
extensively
developed
using
the
CPMV
platform. CPMV particles have
= 3 symmetry and are assembled from
60 copies of each the S and L proteins. The S and L proteins form the so-
called asymmetric unit, and 60 copies of the asymmetric unit form a virion
(Lin
P
, 1999). In a proof-of-concept study, Lys reactivity was probed using
fluorescein NHS ester and isothiocyanate derivatives (Fig. 4.9) Successful
labeling was achieved under forcing conditions (long reaction time and
large excess of labeling agents) where up to 240 labels could be attached
(Wang
et al.
et al.
, 2002a,c).
Figure 4.9
Attachment scheme for attachment of fluorescein NHS (1) and
isothiocyanate (2) derivatives to surface Lys side chains on CPMV. Reproduced with
permission from Wang, Q., Kaltgrad, E., Lin, T. Johnson, J.E., and Finn, M.G. (2002)
Natural supramolecular building blocks: wild-type cowpea mosaic virus,
Chem. Biol.
,
9
(7), 805-811.
In a follow-up study, the nature of the CPMV Lys-reactivity was addressed
in greater detail. The X-ray structure and coordinates of CPMV [available at
the Virus Particle ExploreR (VIPER) database at http://www.viper.scripps.
edu] indicated the presence of five solvent-exposed Lys per asymmetric unit,
which equates to 300 exposed Lys side chains per CPMV particle (Fig. 4.10)
(Chatterji
, 2004a). To investigate whether each of these Lys side chains
are available for bioconjugation, the Lys side chains were systematically
deleted using side-directed mutagenesis. Lys side chains were changed into
Arg side chains. The polarity and positive charge of the Arg side chains are
likely to preserve the microenvironment of the original Lys residues, but
are not expected to engage in the bioconjugation reactions. Unlike Lys, Arg
side chains are poor nucleophiles. The theoretical p
et al.
K
of Arg side chain lies
a
Search WWH ::
Custom Search