| Autor: |
B, Geierstanger, M, Jamin, B F, Volkman, R L, Baldwin |
| Rok vydání: |
1998 |
| Předmět: |
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| Zdroj: |
Biochemistry. 37(12) |
| ISSN: |
0006-2960 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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