Protonation behavior of histidine 24 and histidine 119 in forming the pH 4 folding intermediate of apomyoglobin. Biochemistry 1998 Mar 24;37(12):4254-65
Date
05/02/1998Pubmed ID
9521748DOI
10.1021/bi972516+Abstract
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.
Author List
Geierstanger B, Jamin M, Volkman BF, Baldwin RLAuthor
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApoproteins
Carbon Isotopes
Histidine
Hydrogen-Ion Concentration
Male
Models, Molecular
Mutagenesis, Site-Directed
Myoglobin
Nuclear Magnetic Resonance, Biomolecular
Phenylalanine
Protein Folding
Protons
Valine
Whales
