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at around 12.0. At physiological pH (the pH at which the coupling reactions
were conducted), the guanidine group is protonated and thus a poor
nucleophile and not reactive toward NHS esters or isothiocyanates.
Single, double, triple, and quadruple Lys-minus mutants were generated
and chemical labeling efficiency measured. The studies indicated that all of
the five Lys are indeed available for bioconjugation, and the degree of labeling
efficiency varies between the different sites. The most reactive groups were
found to be Lys 38 on the S protein and Lys 99 on L (Chatterji
et al.
, 2004a).
Figure 4.10
The structure of the viral capsid and the assymetric unit of CPMV.
On the left is a space-filling model of CPMV capsid. The reference asymmetric unit is
framed, and the symmetry elements are labeled. Small (S) subunits labeled A are in
blue, and the large (L) subunits formed by two domains are in red (B domains) and
in green (C domains). The oval represents a twofold axis, the triangle is a threefold
axis, and the pentagon a fivefold axis. Shown on the right is a ribbon diagram of
the asymmetric unit comprised of three jelly rolls — sandwiches, with surface Lys
residues represented as spheres in cyan. Residue numbers are preceded by 1 if they
are in the small subunit and 2 if they are in the large subunit. Lys138 (residue no.
38) and Lys182 (residue no. 82) are in the A domain, Lys299 and Lys234 are in the C
domain, and Lys2199 is in the B domain. Reproduced with permission from Chatterji,
A., Ochoa, W., Paine, M., Ratna, B. R., Johnson, J. E., and Lin, T. (2004) New addresses
on an addressable virus nanoblock: uniquely reactive Lys residues on cowpea mosaic
virus,
Chem. Biol.
,
11
(6), 855-863.
To demonstrate the level of spatial control and orientation that can be
achieved using a VNP as a template for bioconjugation, NHS ester-activated
nanogold was attached to either Lys 38 on S or Lys 99 on L using the
respective mutants displaying a single reactive site. Structural analysis by
cryo-electron microscopy and image reconstruction confirmed specificity
of the reaction. Moreover, it was found that the different microenvironment
impacted the presentation of the ligand (Fig. 4.11) (Chatterji
,
2004a). To date, this level of control has not been achieved with synthetic
nanomaterials.
et al.
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