Protonation behavior of histidine 24 and histidine 119 in forming the pH 4 folding intermediate of apomyoglobin.
Heteronuclear NMR methods are used to study the protonation of histidine and aspartate residues in the acid-induced unfolding of recombinant sperm whale apomyoglobin. The results are combined with fluorescence and circular dichroism measurements of acid-induced unfolding of wild-type and double mutant (H24V/H119F) proteins. They are consistent with a simple model in which the failure to protonate a single buried histidine, H24, is largely responsible for the partial unfolding of native (N) wild-type apomyoglobin to the pH 4 folding intermediate (I). H24 is known to form an unusual interaction in which its side chain is buried and hydrogen-bonded to the side chain of H119. Two-dimensional 1H-15N heteronuclear NMR spectra indicate that H24 is present in the rare delta tautomeric form and remains neutral until N unfolds to I, while H119 becomes protonated before the N --> I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N --> I reaction occurs at lower pH. Therefore, the protonation of aspartate and/or glutamate residues must provide an additional driving force for the N to I reaction. Two-dimensional 1H-13C NMR experiments are used to measure the protonation of aspartates in selectively 13C-labeled apomyoglobin; the results indicate that none of the aspartate residues has a strongly depressed pKa in N, as would be expected if it forms a stabilizing salt bridge.
Geierstanger
B
B
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
Jamin
M
M
Volkman
B F
BF
Baldwin
R L
RL
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000235:genetics; D017510:Protein Folding; D011522:Protons; D014633:Valine / Q000235:genetics; D014907:Whales
D001059:Apoproteins; D002247:Carbon Isotopes; D009211:Myoglobin; D011522:Protons; C010857:apomyoglobin; D010649:Phenylalanine; D006639:Histidine; D014633:Valine