Wards in the keyway: amino acids with anomalouspkas in calycins

As a follow-up to our recent analysis of the
electrostatics of bovine b-lactoglobulin (Eberini et al. in
Amino Acids 42:2019–2030, 2011), we investigated whether
the occurrence in the native structure of calycins—the
superfamily to which b-lactoglobulin belongs—of amino
acids with anomalous pKas is an infrequent or, on the
contrary, a common occurrence, and whether or not a
general pattern may be recognized. To this aim, we randomly
selected four calycins we had either purified from
natural sources or prepared with recombinant DNA technologies
during our previous and current structural and
functional studies on this family. Their pIs vary over several
pH units and their known functions are as diverse as
carriers, enzymes, immunomodulators and/or extracellular
chaperones. In our survey, we used both in silico prediction
methods and in vitro procedures, such as isoelectric
focusing, electrophoretic titration curves and spectroscopic
techniques. By comparing the results under native conditions
(no exposure of the proteins to chaotropic agents) to
those after protein unfolding (in the presence of 8 M urea),
a shift is observed in the pKa of at least one amino acid per
protein, which results in a measurable change in pI. Three
types of amino acids are involved: Cys, Glu, and His, their
position varies along the calycin sequence. Although no
common mechanism may thus be recognized, we hypothesize
that the ‘normalization’ of anomalous pKas may be
the phenomenon that accompanies, and favors, structural
rearrangements such as those involved in ligand binding by
these proteins. An interesting, if anecdotal, validation to
this view comes from the behavior of human retinol
binding protein, for which the pI of the folded and liganded
protein is intermediate between those of the folded and
unliganded and of the unfolded protein forms. Likewise,
both solid (from crystallography) and solution state (from
CD spectroscopy) data confirm that the protein undergoes
structural rearrangement upon retinol binding.