Effective concentrations of amino acid side chains in an unfolded protein.
Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)
Muthukrishnan
K
K
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Nall
B T
BT
D000596:Amino Acids / Q000032:analysis*; D003574:Cytochrome c Group / Q000032:analysis; D007093:Imidazoles / Q000032:analysis; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000596:Amino Acids; D003574:Cytochrome c Group; D007093:Imidazoles; D008024:Ligands; D011522:Protons