The effects of Ca2+ binding on the conformation of calbindin D-28K: A nuclear magnetic resonance and microelectrospray mass spectrometry study
Calbindin D-28K is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D-28K structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D-28K we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D-28K. The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i) apo-calbindin D-28K has an ordered structure which changes to a notably different ordered conformation upon Ca2+ loading, (ii) calcium binding is a sequential process and not a simultaneous event, and (iii) EF-hands 1, 3, 4, and 5 take up Ca2+, whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D-28K has characteristics of both a calcium sensor and a buffer. (C) 2003 Elsevier Science (USA). All rights reserved.