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A Cell‐free Investigation of the Relationship between Myoglobin Expression, Globin Stability and Heme Affinity
Author(s) -
Samuel Premila,
Phillips George,
Olson John
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.882.8
Subject(s) - myoglobin , globin , heme , chemistry , biochemistry , hemeprotein , biophysics , hemoglobin , microbiology and biotechnology , biology , enzyme
We have successfully developed a wheat germ extract cell‐free assay to analyze the relationship between myoglobin (Mb) expression levels and both globin stability and heme affinity. A major advantage of this assay over measurements of Mb production in E. coli is the decoupling of cellular homeostasis with protein expression. Mb variants were selected to include highly stable Mbs of deep diving mammals, unstable Mbs of terrestrial mammals, and Mb mutants with apolar mutations in the heme pocket. Based on our in vitro assay, there is a strong linear correlation between the quantified in vitro expression levels of fully folded Mb variants and their corresponding apoMb unfolding parameters measured independently with purified proteins. ApoMb stability was more critical for holoMb expression than heme affinity. Higher expression levels were observed for Mb mutants with heme pocket apolar mutations that significantly increase apoMb stability but decrease heme affinity. These results confirm previous qualitative and anecdotal studies of Mb expression in E. coli by both Hargrove et al ((1994) Biochemistry 33, 11767–11775) and Scott et al. ((2001) J. Biol. Chem., 275, 27129‐27136) and the conclusions about higher muscle Mb concentrations in deep diving mammals by Mirceta et al ((2013) Science 14, 1324‐1327). Our main goal is to implement this assay for high throughput library screening of globins for enhanced stability. The results of the screens will then be used to engineer more robust recombinant hemoglobins that could potentially be used as oxygen carriers in transfusion medicine. The assay could also be developed for engineering the stability of other proteins. Supported by Grants C‐0612 from the Robert A. Welch Foundation (JSO and PPS) and NIH P01‐HL110900 (JSO).