Structure/Function in the CD site of parvalbumin : understanding calcium affinity using synthetic single site EF-hand peptides

• Main Author: Franchini, Patrick Lorenzo Angelo
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/9968

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.