| Summary: | This study examined the influence of the flanking helices and non-chelating loop
residues in the CD site of carp parvalbumin 4.25 (PCD) on calcium ion (Ca²⁺) affinity
using synthetic single site EF-hand peptide chimeras based on the sequences of PCD and
bovine brain calmodulin site III (Cam3). The peptides are observed to dimerize [Shaw,
G.S., Hodges, R.S., and Sykes, B.D. (1990) Science 249, 280-283.] and a mathematical
model was developed that described the Ca binding process taking into account
dimerization. The model PCD site had 105-fold lower Ca²⁺ affinity than the native site
and did not bind magnesium ions. A Glu to Asp replacement in the -X position of the
Ca²⁺ binding loop in the PCD model site increased Ca²⁺ affinity suggesting repulsion is a
factor in the low Ca²⁺ affinity of the PCD model site. The N-terminal PCD helix gave
higher Ca²⁺ affinity than the helix from Cam3 in both the monomer and dimer forms
possibly through alterations in the N-terminal helix dipole. The C-terminal PCD helix
gave higher Ca²⁺ affinity in the monomer forms and increased monomer negative mean
residue ellipticity. This higher Ca²⁺ affinity may be due to enhanced PCD C-terminal
helix structure. Both the Cam3 N-terminal helix and the PCD C-terminal helix promoted
dimerization possibly through electrostatic interactions. The Gin in non-chelating loop
position 2 from PCD does not alter dimerization or Ca²⁺ binding to the monomer or
dimer forms compared to Lys. The Lys found in PCD loop position 4 appears to have
negative effects on Ca²⁺ affinity in the monomer and dimer forms compared to Gly
possibly through restrictive phi/psi angles or a decrease in the negative charge density in
the loop. The Glu and Asp residues found in position10 and 11 of the PCD loop
promote Ca²⁺ affinity over Ala. This increase may be the result of increased negative
charge density in the loop or increased C-terminal helix stability. Preliminary NMR
studies support the contention that the PCD model site peptide is a dimer in solution.
X-ray diffraction data from a PCD model site peptide crystal has been collected to 1.7 Å
resolution.
|
|---|
