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Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca(2+)-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-([2-[bis(carboxymethyl)amino]-5-methylphenoxy]-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840 nM in the absence and the presence of 150 mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3 nM. Stopped-flow experiments have shown that the dissociation of bound Ca(2+) occurs in two phases, with rates of approx. 1.1 and 0.026 s(-1) corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca(2+) dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca(2+) from DFPase. The Ca(2+) dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca(2+) dissociation and the denaturation results we conclude that the higher-affinity Ca(2+) site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30-40 kDa to overcome problems arising from rapid (1)H and (13)C transverse relaxation. However, (1)H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D(2)O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H(2)O. As a side-effect partial C(alpha) protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary (1)H(N)- and (1)H(alpha)-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D(2)O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C(alpha) protonation of individual amino acids is determined quantitatively and transverse relaxation properties of (1)H(N) and (15)N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.