Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

• Published in: The Journal of biological chemistry 2015-09, Vol.290 (39), p.23479-23495
• Main Authors: Samuel, Premila P., Smith, Lucian P., Phillips, George N., Olson, John S.
• Format: Article
• Language: English
• Subjects: Animals; apomyglobin; bacterial expression of myoglobin; Cell-Free System; cell-free transcription and translation; Escherichia coli - genetics; heme; heterologous expression of globins; myoglobin; Myoglobin - chemistry; Myoglobin - genetics; Myoglobin - metabolism; protein expression; protein folding; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Stability; Protein Structure and Folding
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.672204

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. Background: Myoglobin expression is highly variable in animal tissues and in E. coli. Results: The parameters governing holomyoglobin expression were determined experimentally. Conclusion: Both cell-free and in vivo holomyoglobin yields depend quantitatively on the stability of apoglobin and not hemin affinity. Significance: Enhanced apomyoglobin stability is required for high levels of holoprotein expression due to rapid rates of aggregation of unfolded apoproteins.