Measurement of Ca2+-Binding Constants of Proteins and Presentation of the CaLigator Software

✍ Scribed by Ingemar André; Sara Linse

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 316 KB
• Volume: 305
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5661

✦ Synopsis

The complexity of Ca 2؉ cell signaling is dependent on a plethoria of Ca 2؉ -binding proteins that respond to signals in different ranges of Ca 2؉ concentrations. Since the function of these proteins is directly coupled to their Ca 2؉ -binding properties, there is a need for accurately determined equilibrium Ca 2؉ -binding constants. In this work we outline the experimental techniques available to determine Ca 2؉ -binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for leastsquare fitting directly to the measured quantity. The use of the software is illustrated for Ca 2؉ -binding data obtained for two deamidated forms of calbindin D 9k , either an isospartate-56 (␤ form) or a normal Asp-56 (␣ form). Here, the Ca 2؉ -binding properties of the two isoforms have been studied using the chelator method. The ␣ form shows similar Ca 2؉ -binding properties to the wild type while the ␤ form has lost both cooperativety and affinity.