A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR.
Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.
Tsuge
H
H
Life Science Research Laboratory, Japan Tabacco, Inc., Kanagawa.
Koseki
K
K
Miyano
M
M
Shimazaki
K
K
Chuman
T
T
Matsumoto
T
T
Noma
M
M
Nitta
K
K
Sugai
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D005260:Female; D006736:Horses; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008892:Milk / Q000201:enzymology; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons
D000596:Amino Acids; D001051:Apoenzymes; D011522:Protons; D009113:Muramidase; D002118:Calcium