A photo-chemically induced dynamic nuclear polarization NMR study on rabbit and bovine cytochrome b5.
Although it has been indicated that proteins with chromophoric groups are not suitable for photo-chemically induced dynamic nuclear polarization (photo-CIDNP) measurements, we have successfully obtained these spectra for a heme protein, cytochrome b5. The characteristics of the spectra differed in some points from those so far reported. The intensities of the signals in the aromatic region were very weak, while those of the beta-methylene protons of one histidine and one tryptophan were extremely strong in comparison with the aromatic protons. It was demonstrated, on the basis of the photo-CIDNP spectrum, that one of seven histidines, all three tyrosines and a single tryptophan of the rabbit soluble cytochrome b5 are exposed on the surface of the protein. The results of comparison of the photo-CIDNP spectra for the rabbit soluble and intact, and bovine intact, cytochrome b5 led us to the conclusion that the conformation of the hydrophilic, catalytic part of cytochrome b5 is quite similar among these three proteins. In the presence of Chaps micelles, bovine intact cytochrome b5 was in monomeric form and the histidine signals disappeared from its photo-CIDNP spectrum. When bovine intact cytochrome b5 was reconstituted into egg yolk phosphatidylcholine liposomes, although separate signals due to the protein part were observed in the normal 1H-NMR spectrum, no photo-CIDNP signal could be detected. The normal spectrum suggests that the conformation of the protein embedded in liposomes is similar to that of the oligomeric form without lipids or a detergent.
Hori
A
A
Institute for Protein Research, Osaka University, Japan.
Hayashi
F
F
Kyogoku
Y
Y
Akutsu
H
H
D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group / Q000032:analysis / Q000528:radiation effects*; D015786:Cytochromes b5; D006639:Histidine / Q000032:analysis / Q000528:radiation effects; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D010777:Photochemistry; D011487:Protein Conformation; D011817:Rabbits; D013045:Species Specificity
D003573:Cytochrome b Group; D006639:Histidine; D015786:Cytochromes b5