3.2. Structural features
The ca. 400 kDa native PV2 is an octamer of 4 identical 98 kDa
heterodimers, each composed of one 67 and one 31 kDa subunits. The
presence of intercatenary disulfide bonds among PV2 subunits was
revealed through SDS-PAGE with and without β-mercaptoethanol.
Fig. 4D shows that the 98 kDa heterodimer subunits are held together
by disulfide bridges and that these heterodimers are assembled into
native PV2 by non-covalent forces.
3.3. Proteinase K susceptibility
To analyze the relative exposure of the protein subunits to the
aqueous medium, their susceptibilities to limited proteolysis by
proteinase K were assayed. Incubation of PV2 with the protease,
followed by SDS-PAGE analysis showed extensive degradation of the
67 kDa subunit (Fig. 5). However, the 31 kDa subunit was resistant to
cleavage by protease under controlled conditions. This indicates that
the 31 kDa subunit is not exposed to the aqueous medium or folded in
such a way that it is not susceptible to proteolysis.
3.4. Spectroscopy
The UV absorption spectrum of native PV2, together with its fourth
derivative spectrum is shown in Fig. 4A. It is clearly dominated by
strong signals arising from Trp (290 nm) and Phe (260 nm),
respectively. The fluorescence emission spectrum of the protein is
shown in Fig. 4B where a maximum at 338 nmsuggests that Trp indole
rings are placed in a hydrophobic environment, also indicated by CD
and the acrylamide fluorescence quenching experiments (see below).
The fCD and nCD spectra obtained for native PV2 are depicted in
Fig. 4C. Both the K2D algorithm [20] and CONTIN algorithm [21]
applied to the fCD data gave consistent results estimating an average of
21% α structure, 25% β structure and 54% random coil or unstructured