Heterologous Overexpression of Human NEFA and Studies on the Two EF-Hand Calcium-Binding Sites
Autor: | Stefan Otte, Hartmut Kratzin, Norbert Hilschmann, Shitsu Barnikol-Watanabe, Barnikol Hu, Hilde Götz, Katja A. Kroll, Hans Sternbach, Gregor Hirschfeld |
---|---|
Rok vydání: | 1999 |
Předmět: |
Circular dichroism
Conformational change Time Factors Biophysics chemistry.chemical_element Nerve Tissue Proteins Calcium Biochemistry Pichia Pichia pastoris NEFA Humans Nucleobindins Trypsin Binding site Molecular Biology Binding Sites Dose-Response Relationship Drug Models Genetic biology EF hand Circular Dichroism Calcium-Binding Proteins Proteolytic enzymes Genetic Variation Cell Biology biology.organism_classification DNA-Binding Proteins chemistry Mutagenesis |
Zdroj: | Biochemical and Biophysical Research Communications. 260:1-8 |
ISSN: | 0006-291X |
DOI: | 10.1006/bbrc.1999.0867 |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |