Heterologous overexpression of human NEFA and studies on the two EF-hand calcium-binding sites

Human NEFA is an EF-hand, leucine zipper protein containing a signal sequence. To confirm the calcium binding capacity of NEFA, recombinant NEFA analogous to the mature protein and mutants with deletions in the EF-hand domain were expressed in Pichia pastoris and secreted into the culture medium at high yield. The calcium binding activity of each purified protein was measured by a modified equilibrium dialysis using the fluorescent Ca2+ indicator FURA-2 and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/mol NEFA was determined. The Ca2+ binding constants were resolved by intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca2+ binding sites with Kd values of 0.08 microM and 0.2 microM. Circular dichroism (CD) spectroscopy showed an increase from 30 to 43% in the amount of alpha-helix in NEFA after addition of calcium ions. Limited proteolytic digestion indicated a Ca2+ dependent conformational change accompanied by an altered accessibility to the enzyme.