Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf.
We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.
Guiles
R D
RD
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Altman
J
J
Kuntz
I D
ID
Waskell
L
L
Lipka
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015786:Cytochromes b5; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011817:Rabbits; D012995:Solubility; D013552:Swine; D014357:Trypsin
D015786:Cytochromes b5; D014357:Trypsin