Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Ca 2+ -Binding Proteins Using ESI-MS 163
As stated earlier, the nature of the measurements obtained with ESI-MS
yields precise information about stoichiometry. This is illustrated by an ESI-MS
study of the EF-hand protein calbindin D 28K, which has been shown using to
bind between three and six calcium ions by various solution methods. ESI mass
spectra showed clearly that four calcium ions are bound to calbindin D 28K as a
mass difference of 151 Da (expected = 4 × 40 Da (mass of Ca 2+ )– 4 × 2 Da
(loss of protons) = 152 Da) was observed between the apo and the holo forms
of the protein. Mutants that contained deletions of one or two of the EF hands
were used to help localize the sites of metal binding (9).
A recent study of human parvalbumin provides a good example of sensitivity
to structural modifications. Variants that had mutations in both of the two
EF hands did not bind any Ca 2+ acting as a suitable negative control and indicating
that the interactions observed between the parvalbumins and calcium
were indeed specific. Wild-type protein bound two Ca 2+ , whereas the proteins
that had mutations in one EF hand bound one calcium (10). This method was
also used to study parvalbumin extracted from murine and rat tissue samples
(10). Additional calcium-binding proteins for which Ca 2+ stoichiometry has
been determined by ESI-MS include parvalbumin (3,5,11), lactalbumin (5),
matrylisin (12), and the stromelysin catalytic domain (5).
ESI-MS has been used to study cooperativity of binding and to compare
binding affinity for different metals ions. Typically, a metal-free protein is
titrated with a metal ion and the ESI mass spectra are obtained at various metal
concentrations. Examination of the relative amounts of co-existing species at
each concentration yields information about preferential binding. For example,
if a protein has two Ca 2+ -binding sites that are both high affinity and independent,
the mass spectrum should reveal the presence of the mixture apo-protein:
Ca 2+ -protein: (Ca 2+ ) 2-protein in a 1:2:1 ratio when one equivalent of Ca 2+ (with
respect to the protein) is present. If the sites were independent, but one had a
higher affinity, the mass spectrum should primarily show a species with only
one Ca 2+ bound. Alternatively, if the binding was positively cooperative, one
would expect that the spectrum would show some apo-protein and (Ca 2+ ) 2protein
with little (Ca 2+ ) 1-protein present. This is illustrated in a study of the
binding of Ca 2+ and Cd 2+ to wild-type and mutant calbindin D 9K (13). The
spectrum of the wild-type protein indicates that as the calcium concentration is
increased, the calbindin proceeds from being in the apo form to binding
two Ca 2+ with the (Ca 2+ ) 1-species present in very small amounts. The mutant
N56A calbindin, when titrated with calcium acetate, shows high amounts of
apo-calbindin, (Ca 2+ ) 1-calbindin and (Ca 2+ ) 2-calbindin depending on the Ca 2+
concentration. This type of spectrum was also observed for the binding of Cd 2+
to both the wild-type and mutant proteins. Thus, wild-type calbindin D 9K binds