Proton nuclear magnetic resonance studies of porcine intestinal calcium binding protein

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Abstract
1H NMR was employed to study the environment of several proton nuclei (primarily those arising from aromatic residues) of the porcine intestinal Ca binding protein. An assignment for the single tyrosine (Tyr-16) residue was made on the basis of laser photochemically induced dynamic nuclear polarization (CIDNP) and homonuclear decoupling experiments. pH titration studies have shown that the tyrosine pKa is unusually high in the apoprotein and increases even further upon the addition of Ca. However, the observation of a CIDNP effect with this tyrosine in both the presence and absence of Ca indicates that it is solvent accessible and therefore exposed on the surface of the molecule. Under the conditions of these experiments, the protein was observed to bind Ca with a 2:1 stoichiometry, at a rate of exchange slow enough that the NMR spectra are in the slow-exchange limit. The presence of upfield shifted phenylalanine and methyl resonances in the apoprotein indicates that there is a well-defined tertiary structure in the absence of Ca.

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