An in Vitro Investigation of Globin Folding and Expression

Abstract

We developed a globin expression cell-free assay to analyze the relationship between myoglobin stability and expression levels. Major advantages of this assay over measurements of globin expression in vivo include the decoupling of cellular homeostasis with protein expression and the ability to control various transcription and translation variables. Myoglobin variants in our study were selected to include highly stable myoglobins of deep diving mammals, unstable myoglobins of terrestrial mammals, and myoglobin mutants with heme pocket apolar mutations that significantly increase apomyoglobin stability but decrease heme affinity. Surprisingly, higher expression levels were also observed for these myoglobin mutants, demonstrating a critical compromise for expression. The results from this assay show a strong linear correlation between in vitro expression levels of fully folded holomyoglobin variants and their corresponding apomyoglobin unfolding parameters measured independently with purified proteins (Samuel et al. (2015) J. Biol. Chem. 290, 23479-23495). These results confirmed previous qualitative and anecdotal studies of myoglobin expression in E. coli and the conclusions about higher muscle Mb concentrations in deep diving mammals by (Mirceta et al. (2013) Science 14, 1324-1327). Our more recent theoretical modeling of expression shows that high globin stability is critical for efficient holomyoglobin formation to overcome the high rates of aggregation and precipitation that occur when large amounts of the apoglobin are translated rapidly. These analyses and methods are being extended to the more complex globin folding and expression mechanisms for native and recombinant hemoglobin tetramers, which are composed of subunits that are structurally similar to myoglobin.