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J. Biol. Chem., Vol. 275, Issue 35, 27129-27136, September 1, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/35/27129    most recent M000452200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Scott, E. E. Articles by Olson, J. S. Search for Related Content PubMed PubMed Citation Articles by Scott, E. E. Articles by Olson, J. S. Social Bookmarking                      What's this?

The Stabilities of Mammalian Apomyoglobins Vary over a 600-Fold Range and Can Be Enhanced by Comparative Mutagenesis^*

Emily E. Scott, Eden V. Paster, and John S. Olson

From the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892

Apomyoglobins from 13 different mammals were examined for resistance to denaturation by guanidinium chloride. Unfolding was followed by circular dichroism and tryptophan fluorescence and analyzed globally using the two-step, three-state mechanism first described by Barrick and Baldwin (Barrick, D., and Baldwin, R. L. (1993) Biochemistry 32, 3790-3796). With one exception, the rise and fall of Trp fluorescence intensity correlates quantitatively with the native to intermediate to unfolded steps seen in the CD curves. Although the O₂ binding properties of the holoproteins are nearly identical, the unfolding transitions of the apomyoglobins show 600-fold differences in resistance to guanidinium chloride denaturation. Apomyoglobins from diving mammals, particularly from sperm whales, are the most stable, whereas the apoproteins from pig, horse, and sheep are the least stable, indicating selective pressure for resistance to denaturation in the whale proteins. Sequence comparisons suggest that the key stabilizing residues in whale globins are Ala⁵, His¹², Ile²⁸, Thr⁵¹, Ala⁵³, Ala⁷⁴, Lys⁸⁷, Lys¹⁴⁰, and Ile¹⁴². Combinations of these residues were substituted into pig myoglobin. The resultant multiple mutants showed stabilities approaching that of recombinant sperm whale apomyoglobin. Thus, comparative mutagenesis can be used to increase heme protein stability and improve expression yields in bacteria without compromising function.

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