The effects of Ca(2+) 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 Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), 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.