Protonation behavior of histidine 24 and histidine 119 in forming the pH 4 folding intermediate of apomyoglobin. Biochemistry 37:4254–4265

• Main Authors: Bernhard Geierstanger, Marc Jamin, Brian F. Volkman, Robert L. Baldwin
• Other Authors: The Pennsylvania State University CiteSeerX Archives
• Format: Text
• Language: English
• Published: 1998
• Subjects: Sperm whale
• Online Access: http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.664.2854; http://rbaldwin.stanford.edu/PDFs/protonation_behavior_of_histidine.pdf

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 ä tautomeric form and remains neutral until N unfolds to I, while H119 becomes protonated before the N f I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N f 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.