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.
Kroll
K A
KA
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Strasse 3, Göttingen, D-37075, Germany. kroll@exmdi1.gwdg.de
Otte
S
S
Hirschfeld
G
G
Barnikol-Watanabe
S
S
Götz
H
H
Sternbach
H
H
Kratzin
H D
HD
Barnikol
H U
HU
Hilschmann
N
N
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D014644:Genetic Variation; D006801:Humans; D008957:Models, Genetic; D016296:Mutagenesis; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010843:Pichia / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C000630650:NUCB2 protein, human; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D014357:Trypsin; D002118:Calcium