Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.
The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Aramini
J M
JM
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Drakenberg
T
T
Hiraoki
T
T
Ke
Y
Y
Nitta
K
K
Vogel
H J
HJ
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002127:Calcium Isotopes; D002417:Cattle; D010856:Columbidae; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008962:Models, Theoretical; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
D002127:Calcium Isotopes; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium