The effect of sequence truncation and amino-acid substitutions on the biophysical properties of protein S and calbindin D9k

Abstract: Protein S is a modular protein and a cofactor in the protein C anticoagulant system. It consists of a Gla-module, a thrombin-sensitive loop, four EGF-modules and a SHBG-like module. Protein S has previously been shown to contain four binding sites with high affinities for Ca2+ (Ka>9x105 M-1 at physiological ionic strength) and at least one of the sites has been located to a fragment containing the third and the fourth EGF-module. In this thesis, the Ca2+ affinity of the third and fourth EGF-module as isolated peptides has been determined using 1H-NMR spectroscopy. At physiological pH and ionic strength, each module was found to have a single Ca2+-binding site with low affinity (Ka=1.6x102 M-1 for the third and 1.2x102 M-1 for the fourth EGF module). The same sites in protein fragments containing EGF 1-4 or EGF 2-4 were in a related study found to have 103-104-fold higher affinities, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. Calbindin D9k is a mammalian Ca2+-binding protein with Ca2+-transport/buffer functions. It consists of two EF-hands and is one of the smallest members of the ubiquitous EF-hand protein family. The effects of hydrophobic core mutations on the stability of calbindin D9k towards urea unfolding were determined using CD spectroscopy. Eleven mutations involving eight residues were examined. The mutations were found to exert large effects on the stability with denaturation midpoint concentrations, CM, ranging from 1.8 to 6.6 M urea and deltaGNU(H2O) ranging from 6.6 to 27.4 kJ/mol. The results were compared with similar studies performed on other proteins and a correlation was found between the change in free energy of unfolding and the absolute change in side-chain volume upon mutation. This means that a mutation involving increase in side-chain volume on average is as destabilizing as a volume decrease. The large range in stabilities for the mutants of this study made it difficult to accurately determine the baselines of the CD intensity for the native and unfolded states of some of the mutants. This problem is discussed and a general solution is proposed. Isolated EF-hand peptides have in several cases been found to dimerize in the presence of Ca2+. The coupled homo-dimerization and Ca2+-binding equilibria of the two EF-hands of calbindin D9k have been investigated using series of Ca2+-titrations performed at different peptide concentrations. The titrations were monitored using CD and fluorescence spectroscopy. We found that for the first EF-hand, Ca2+-binding and dimerization was coupled, but the second EF-hand did not dimerize even in the presence of Ca2+. The effect of hydrophobic amino-acid substitutions on the reconstitution of calbindin D9k from EF-hand fragments was studied using surface plasmon resonance. Six substitutions at four sites in the second EF-hand were studied. A strong correlation between the change in dissociation constant and the change in stability of the calcium form of the intact protein was found, indicating that the hydrophobic effect is important mainly for the interactions between the two sub-domains.