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dependence of this interaction on the solution pH and ionic strength
[10-14], we went on to perform electrostatic calculations in an effort
to understand the nature of the C3d/CR2 association at a theoretical
level [15].
This was achieved by analyzing the overall electrostatic field of each
protein both in free form and in complex, and also by measuring the
contribution of charged residues within each protein to the binding
reaction [15]. To gain further insight into the involvement of individual
C3d residues to the C3d/CR2 association, theoretical site-directed
mutations were designed [15] on the basis of available crystallographic
structures, and their electrostatic potentials were subsequently calcu-
lated. It should be noted that these theoretical calculations showed
great correlation to the experimental data produced by in vitro muta-
genesis studies [12], and supported a two-step association model
comprising recognition and binding [15]. An integrative survey
addressing the complex dynamics and biophysical nature of the
C3d/CR2 interaction should provide a comprehensive platform for the
design of more potent complement therapeutics.
Interestingly, a similar biophysical approach was recently employed
to determine the role of electrostatic potential in predicting the func-
tional activity of two viral complement inhibitory proteins, VCP and
SPICE [16]. These two orthopoxvirus complement regulators differ only
in 11 amino acids. Yet, SPICE exhibits a 100-fold more potent inhibitory
activity on complement [17]. Electrostatic modeling clearly predicted
that switching critical residues between these two viral proteins would
result in a VCP protein with enhanced complement regulatory activity.
Furthermore, this study concluded that electrostatic forces are a major
determinant of VCP binding to C3b, suggesting a two-step association
model that involves electrostatically driven recognition and enhanced
binding, as in the case of the C3d/CR2 association.
Mass Spectrometry and Hydrogen/Deuterium Exchange Studies
Hydrogen/deuterium exchange has traditionally been used to under-
stand the formation of protein core or stable intermediate or transient
states in pathways of protein folding, because it provides a noninvasive
method for identifying protected (or deprotected) exchanging amides.
The same principles can be applied to studies of protein-protein
association, where the loss in solvent-accessible surface area upon asso-
ciation can be correlated with amide protection from exchange for the
amides that lose their contact with solvent. Recent advances in the
use of mass spectrometry allow for rapid collection of data of free and
complexed proteins [18-20]. Comparison of mass spectra of free and
complexed proteins provides the sites of interaction without the need
for previously available structural data. The efficiency and rapidity of the
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