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

Abstract

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.

Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase

“…The heme insertion into apo-Mb is blocked by hsp90 inhibitors and is stimulated when cells contain an active sGC-cGMP signaling pathway. be particularly useful in elucidating the molecular mechanisms of hsp90-driven client maturation because its structural folding-unfolding transitions are robust and among the most well-studied in biology (56)(57)(58). Work with Mb could also guide investigations of how hsp90 aids maturation of more complex globins like Hb-b and g, which unlike Mb form mixed tetrameric structures with an Hb-a partner during maturation.…”

Lessons Learned from 50 Years of Hemoglobin Research: Unstirred and Cell-Free Layers, Electrostatics, Baseball Gloves, and Molten Globules

“…Hb folding and assembly with heme. These schemes were taken from Samuel et al (90,91) and are described in the text. 37,49,55,58,62,98).…”

“…The second step involves folding of most of the heme pocket to generate the final native apoMb state, N, in which only the F-helix and parts of the C-helix and CD corner are disordered. This mechanism is strongly supported by unfolding studies in which mutations that fill the heme pocket with large apolar amino acids (i.e., His(E7) to Leu or Phe, Val(E11) to Phe, Leu(B10) to Phe) greatly stabilize the N state, whereas mutations that introduce more polar residues (i.e., Val(E11) to Thr or Asn, Leu(B10) to Asn) destabilize the N state and facilitate formation of the molten globule intermediate (37,91).…”

Tracking evolution of myoglobin stability in cetaceans using experimentally calibrated computational methods that account for generic protein relaxation

“…Thus, a conspicuous relationship between experimentally observed elevations in Mb stability and concentration can be rationalized and argues for selection against misfolded protein copies via protein stability [1]. 6 Heterologous expression levels of Mb orthologs are also proportional to their respective ApoMb stabilities but depend little or even inversely on heme affinity, because high ApoMb stability increases the life time and thus total copy number of holoMb formed upon heme incorporation [24], which is a specific event on the folding pathway of almost completely folded ApoMb [25]. This leads to an apparent correlation between heterologous expression levels and Mb concentrations measured in the real host mammals; in both cases, this arises from the stability of the Mb ortholog[1] [24].…”

“…6 Heterologous expression levels of Mb orthologs are also proportional to their respective ApoMb stabilities but depend little or even inversely on heme affinity, because high ApoMb stability increases the life time and thus total copy number of holoMb formed upon heme incorporation [24], which is a specific event on the folding pathway of almost completely folded ApoMb [25]. This leads to an apparent correlation between heterologous expression levels and Mb concentrations measured in the real host mammals; in both cases, this arises from the stability of the Mb ortholog[1] [24]. However, the dependency on stability arises for different reasons: In one case due to protein synthesis rates, in the other, as argued previously [1], due to selection pressure on steady state concentrations of whales.…”