Human NEFA is an EF-hand, leucine zipper protein containing a signal sequen
ce. To confirm the calcium binding capacity of NEFA, recombinant NEFA analo
gous 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 measu
red by a modified equilibrium dialysis using the fluorescent Ca2+ indicator
FURA-S and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/m
ol NEFA was determined. The Ca2+ binding constants were resolved by intrins
ic fluorescence spectroscopy. Fluorescence titration exhibited two classes
of Ca2+ binding sites with Kd values of 0.08 mu M and 0.2 mu M. Circular di
chroism (CD) spectroscopy showed an increase from 30 to 43% in the amount o
f alpha-helix in NEFA after addition of calcium ions. Limited proteolytic d
igestion indicated a Ca2+ dependent conformational change accompanied by an
altered accessibility to the enzyme. (C) 1999 Academic Press.