The effects of Ca super(2+) binding on the conformation of calbindin D sub(28K): A nuclear magnetic resonance and microelectrospray mass spectrometry study

• Published in: Analytical biochemistry 2003-06, Vol.317 (1), p.59-66
• Main Authors: Venters, R A, Benson, L M, Craig, T A, Paul, KH, Kordys, DR, Thompson, R, Naylor, S, Kumar, R, Cavanagh, J
• Format: Article
• Language: English
• ISSN: 0003-2697
• DOI: 10.1016/S0003-2697(03)00084-8

Calbindin D sub(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 sub(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D sub(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D sub(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i) apo-calbindin D sub(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca super(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 super(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D sub(28K) has characteristics of both a calcium sensor and a buffer.