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

  • Premila P. Samuel
    Affiliations
    BioSciences at Rice, Rice University, Houston, Texas 77005
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  • Lucian P. Smith
    Footnotes
    Affiliations
    BioSciences at Rice, Rice University, Houston, Texas 77005
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  • George N. Phillips Jr.
    Affiliations
    BioSciences at Rice, Rice University, Houston, Texas 77005

    Department of Chemistry, Rice University, Houston, Texas 77005
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  • John S. Olson
    Correspondence
    To whom correspondence should be addressed: BioSciences at Rice, MS 140, Rice University, 6100 Main St., Houston, TX 77005-1892. Tel.: 713-348-4762; Fax: 713-348-5154.
    Affiliations
    BioSciences at Rice, Rice University, Houston, Texas 77005
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant P01 HL110900 (to J. S. O.). This work was also supported by Grant C-0612 from Robert A. Welch Foundation (to J. S. O.). The initial funds for setting up the cell-free technology at Rice University were supported by the National Institutes of Health Grant U01 GM098248 (Protein Structure Initiative) (to G. N. P., Jr.). The earlier work on holoMb expression in E. coli was supported by the National Institutes of Health Grants HL47020 and GM35649 (to J. S. O.). The authors declare that they have no conflicts of interest with the contents of this article.
    ♦ This article was selected as a Paper of the Week.
    1 Present address: Dept. of Bioengineering, University of Washington, Seattle, WA 98195 (consultant).
Open AccessPublished:July 23, 2015DOI:https://doi.org/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.

      Introduction

      Myoglobin (Mb)
      The abbreviations used are: Mb
      myoglobin
      apoMb
      myoglobin with no heme bound
      holoMb
      myoglobin containing bound heme
      Sw
      sperm whale
      Dw
      dwarf sperm whale
      GdnHCl
      guanidine HCl
      N
      native
      U
      unfolded.
      serves as a model system for examining the factors that govern the expression of large amounts of heme proteins for which both protein folding and cofactor binding are required. It is a single domain globin comprised of eight α-helical segments, labeled A to H, that can bind iron protoporphyrin IX either in the ferrous (heme) or ferric state (hemin) through axial coordination of the iron atom with a proximal histidine (His-93 at the F8 helical position). Ligands bind to the iron atom on the distal side of the protoporphyrin ring and include O2 in the ferrous state and H2O in the ferric state. These exogenous ligands are stabilized by hydrogen bonding to the distal histidine (His-64 at the E7 helical position) in almost all wild-type mammalian Mbs (
      • Quillin M.L.
      • Arduini R.M.
      • Olson J.S.
      • Phillips Jr., G.N.
      High resolution crystal structures of distal histidine mutants of sperm whale myoglobin.
      ).
      Recently, Culbertson and Olson (
      • Culbertson D.S.
      • Olson J.S.
      Role of heme in the unfolding and assembly of myoglobin.
      ) developed the six-state model shown in Fig. 1 to describe the unfolding of holo-metMb and dissociation of hemin and then used it to analyze quantitatively sets of GdnHCl-induced folding curves for a series of holo- and apoMb variants. This analysis was based on the mechanisms reported by Barrick and co-workers (
      • Hughson F.M.
      • Barrick D.
      • Baldwin R.L.
      Probing the stability of a partly folded apomyoglobin intermediate by site-directed mutagenesis.
      ,
      • Barrick D.
      • Baldwin R.L.
      Three-state analysis of sperm whale apomyoglobin folding.
      ) and Wright and co-workers (
      • Eliezer D.
      • Yao J.
      • Dyson H.J.
      • Wright P.E.
      Structural and dynamic characterization of partially folded states of apomyoglobin and implications for protein folding.
      ,
      • Nishimura C.
      • Dyson H.J.
      • Wright P.E.
      Identification of native and non-native structure in kinetic folding intermediates of apomyoglobin.
      ) for the unfolding of apoMb and the generation of a molten globule intermediate (I) in which the heme pocket was melted but the A, G, and H helices remained mostly intact. Culbertson and Olson (
      • Culbertson D.S.
      • Olson J.S.
      Role of heme in the unfolding and assembly of myoglobin.
      ) showed that hemin can bind to the intermediate to generate a hemichrome structure but that the affinity of the I state for hemin is much less than that of the native (N) state. In the absence of GdnHCl, unfolding leads to aggregation of the unfolded globin states (UH, U), making the process irreversible. Any released free hemin is highly toxic in vivo where it partitions into membranes and promotes lipid oxidation and generation of reactive oxygen species (
      • Alayash A.I.
      • Patel R.P.
      • Cashon R.E.
      Redox reactions of hemoglobin and myoglobin: biological and toxicological implications.
      ,
      • Alayash A.I.
      • Andersen C.B.
      • Moestrup S.K.
      • Bülow L.
      Haptoglobin: the hemoglobin detoxifier in plasma.
      ,
      • Grunwald E.W.
      • Richards M.P.
      Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish muscle.
      ,
      • Lee S.K.
      • Tatiyaborworntham N.
      • Grunwald E.W.
      • Richards M.P.
      Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate.
      ).
      Figure thumbnail gr1
      FIGURE 1Six-state mechanism for holoMb unfolding modified from Culbertson and Olson (
      • Culbertson D.S.
      • Olson J.S.
      Role of heme in the unfolding and assembly of myoglobin.
      ). The three states of apoMb are native (N) with most of the helices folded, intermediate (I) with the heme pocket mostly unfolded, and completely unfolded (U). HoloMb states containing bound heme (H) are native (NH), intermediate (IH) with the heme pocket melted and a hemichrome structure, and unfolded (UH) with heme bound nonspecifically.
      Most studies of gene expression examine the regulation of mRNA synthesis by promoter sequences and transcription factors, mRNA structure and stability, and rates of translation on ribosomes. Our focus is on the final steps of expression involving the folding and assembly of fully functional holomyoglobin, which include the reverse of the processes shown in Fig. 1 and depend on the amino acid sequence of the polypeptide chain. Unfolded (U) polypeptide comes off the ribosomes, folds into the N state, and binds heme to produce the ferric NH state, which in turn can be reduced, bind O2 or CO, and be further stabilized in the holoprotein form. However, as shown in Fig. 1, net synthesis of holo-metMb and its reduction competes with aggregation and precipitation of both unfolded apoprotein and free hemin. Thus, multiple factors should contribute to expression yields of holoMb, including apoprotein stability, hemin affinity, and reduction and ligand binding.
      Initial semi-quantitative studies by Hargrove et al. (
      • Hargrove M.S.
      • Krzywda S.
      • Wilkinson A.J.
      • Dou Y.
      • Ikeda-Saito M.
      • Olson J.S.
      Stability of myoglobin: a model for the folding of heme proteins.
      ) suggested that heterologous expression of mammalian Mbs in Escherichia coli is governed more by globin stability than by hemin affinity. Scott et al. (
      • Scott E.E.
      • Paster E.V.
      • Olson J.S.
      The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis.
      ) showed that apomyoglobins from deep diving whales are 10–500 times more resistant to unfolding than apoglobins from terrestrial animals. They suggested that the increased resistance to denaturation was required due to acidosis that occurred in whale muscle during prolonged dives. Scott et al. (
      • Scott E.E.
      • Paster E.V.
      • Olson J.S.
      The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis.
      ) also noted that the enhanced apoglobin stability could explain the high expression yields of sperm whale holoMb in E. coli but not pig or human holoMbs. This issue had been puzzling because most mammalian holoMbs appear to have similar stabilities (
      • Hargrove M.S.
      • Krzywda S.
      • Wilkinson A.J.
      • Dou Y.
      • Ikeda-Saito M.
      • Olson J.S.
      Stability of myoglobin: a model for the folding of heme proteins.
      ,
      • Flanagan M.A.
      • Garcia-Moreno B.
      • Friend S.H.
      • Feldmann R.J.
      • Scouloudi H.
      • Gurd F.R.
      Contributions of individual amino acid residues to the structural stability of cetacean myoglobins.
      ). However, Scott et al. (
      • Scott E.E.
      • Paster E.V.
      • Olson J.S.
      The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis.
      ) did not examine the differences in expression of the mammalian Mbs in E. coli quantitatively.
      More recently, in a bioinformatics study, Mirceta et al. (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ) suggested that the enhanced apoprotein stabilities of deep diving mammal Mbs evolved to allow much higher expression of the protein in the muscles of these animals. For example, the level of Mb in the skeletal muscles of sperm whale is roughly 70 mg/g of wet tissue, whereas the amount in pig muscle is only 2–4 mg/g of wet tissue, a greater than 10-fold difference (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ). They noted that MbO2 is the primary source of oxygen for swimming during deep dives when blood circulation is diverted from the skeletal muscles to keep the heart and brain of the diving animal well oxygenated. Mirceta et al. (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ) also discovered that the higher expressing Mbs in aquatic mammals had, on average, a larger positive surface charge (ZMb = +2.5 to +4.8 at neutral pH) that may have contributed to their higher apoglobin stability.
      Between 2001 and 2003, Smith (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ) surveyed the expression levels in E. coli of a large library of ∼250 site-directed and randomly generated heme pocket mutants of sperm whale Mb. All the variant genes were cloned into the same expression vectors, and the goal was to examine compromises between apoglobin expression, stability, and functional O2 binding. Smith (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ) observed a correlation between the overall folding constant (1/KNU or KUN) and the relative expression yield in the E. coli, but the scatter in the data were substantial and there were clear outliers, which suggested enhanced susceptibility to proteolysis (Fig. 10A) (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ).
      Figure thumbnail gr10
      FIGURE 10Heterologous expression of holoMb and correlations with overall apoMb stability. A, in vivo expression of Sw Mb mutants in E. coli relative to expression of wild-type Sw Mb. Gray and black circles represent apoMb unfolding data measured at low (20 mm) and high (200 mm) potassium phosphate concentrations, respectively, at 25 °C, pH 7.0, with 5 μm apoMb. Data were taken from Smith (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ). B, relative expression of mammalian Mbs in the cell-free/wheat germ system. The relative expression values in B were calculated using and are also given in TABLE 1, TABLE 2. Approximate concentration in mg/(g of wet tissue) of native Mbs found in the muscles of terrestrial (4–8 mg/g) and deep diving mammals (40–70 mg/g) are given in parentheses in red and were taken from Mirceta et al. (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ). The apoMb equilibrium unfolding constants for all species except pig (refer to ) were measured with 10 μm protein in 10 mm potassium phosphate, pH 7, at 20 °C. The overall apoMb stability is given by log(KUN), which is proportional to the free energy released during folding from the unfolded to the native state.
      In this work, we have adapted the CellFree Sciences (ENDEXT® technology) in vitro protein synthesis system (
      • Spirin A.S.
      • Swartz J.R.
      ,
      • Takai K.
      • Sawasaki T.
      • Endo Y.
      Practical cell-free protein synthesis system using purified wheat embryos.
      ) to test and examine quantitatively the various ideas about holoMb expression that were derived from our previous more qualitative studies. The decoupled in vitro transcription and wheat germ-based translation system allows more control over the amounts of DNA, mRNA, amino acids, ATP, and hemin present (
      • Spirin A.S.
      • Swartz J.R.
      ). Most proteases have also been eliminated (
      • Takai K.
      • Sawasaki T.
      • Endo Y.
      Practical cell-free protein synthesis system using purified wheat embryos.
      ). In addition, the soluble holoMb product can be quickly separated from the translation mixture and partially purified, and its spectral properties in the ferric state can be measured quantitatively.
      In E. coli most of these variables cannot be as easily controlled. Because the availability of cofactor can also be limiting, hemin is often added exogenously, with or without co-expression of heme transporter genes, or δ-aminolevulonic acid is added to enhance bacterial heme synthesis (
      • Graves P.E.
      • Henderson D.P.
      • Horstman M.J.
      • Solomon B.J.
      • Olson J.S.
      Enhancing stability and expression of recombinant human hemoglobin in E. coli: progress in the development of a recombinant HBOC source.
      ,
      • Varnado C.L.
      • Mollan T.L.
      • Birukou I.
      • Smith B.J.
      • Henderson D.P.
      • Olson J.S.
      Development of recombinant hemoglobin-based oxygen carriers.
      ). In the expression studies performed by Smith (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ) in E. coli, no external hemin was added; Mb was not purified from lysates; and absolute spectra were not measured. Another complicating factor in E. coli is that the expressed holoMb is kept reduced in the bacterial cytoplasm, and in some cases, it binds endogenously produced CO, which could greatly stabilize the protein, particularly those variants in which the distal histidine is replaced with Phe or Leu (
      • Quillin M.L.
      • Arduini R.M.
      • Olson J.S.
      • Phillips Jr., G.N.
      High resolution crystal structures of distal histidine mutants of sperm whale myoglobin.
      ,
      • Olson J.S.
      • Eich R.F.
      • Smith L.P.
      • Warren J.J.
      • Knowles B.C.
      Protein engineering strategies for designing more stable hemoglobin-based blood substitutes.
      ).
      We have used the cell-free protein synthesis system to examine holo-metMb expression for a library of naturally occurring Mb variants, including pig, human, sperm whale, gray seal, goosebeak whale, and dwarf sperm whale, which span the range of in vitro apoglobin stabilities reported by Scott et al. (
      • Scott E.E.
      • Paster E.V.
      • Olson J.S.
      The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis.
      ) and the levels of myoglobin found in skeletal muscle as reported by Mirceta et al. (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ). Gray seal Mb was chosen at the suggestion of Berenbrink and co-workers (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ) based on their sequence comparisons and preliminary unfolding studies, both of which indicate strongly that deep diving seals also have large apoMb stability constants. Berenbrink and co-workers are systematically testing this idea of a convergent evolution of high Mb stability in various families of diving animals.
      M. Berenbrink, personal communication.
      Then, three distal pocket mutants were constructed to significantly enhance globin stability but reduce hemin affinity based on the results described by Smith (
      • Smith L.P.
      The Effects of Amino Acid Substitution on Apomyoglobin Stability, Folding Intermediates, and Holoprotein Expression.
      ) and include: H64F/V68F sperm whale Mb and H64L and H64F/V68F dwarf sperm whale Mb.
      Our study verifies unambiguously that apoMb stability determines holoprotein expression levels, regardless of whether translation occurs in vitro, in E. coli, or in mammalian myocytes (
      • Mirceta S.
      • Signore A.V.
      • Burns J.M.
      • Cossins A.R.
      • Campbell K.L.
      • Berenbrink M.
      Evolution of mammalian diving capacity traced by myoglobin net surface charge.
      ). This work also lays the groundwork for high throughput screening of apoprotein stability for large libraries of globin variants without having to purify milligram quantities of pure protein.

      Author Contributions

      P. P. S. helped design the study, performed all the experiments except those involving expression in E. coli, analyzed all of the data, and helped to write the initial draft of the paper, all as part of her Ph.D. thesis project. G. N. P. Jr. provided the initial idea of using the cell-free system, helped design key parts of the in vitro expression system and its analysis, and edited the final manuscript. L. P. S. did all the previous work on holoMb expression in E. coli, including the results in Fig. 10A, provided the initial idea that expression yield depends almost exclusively on overall apoMb stability, and edited the paper. J. S. O. helped to design the in vitro unfolding and hemin dissociation experiments, analyze the data, derive the theory for rates of expression, write the initial draft of the paper, and edit the final version.

      Acknowledgments

      We thank Eileen W. Singleton, Jayashree Soman, and William Ou for their assistance preparing some of the Mb variants and Pierce Young (Bonnie Bartel laboratory, Rice University) for help with doing Western blots to determine the specificity of the anti-Mb antibody used in the ELISA. We acknowledge Emily T. Beebe, Russell L. Wrobel, and Shin-ichi Makino from University of Wisconsin-Madison for providing assistance in establishing the cell-free methods at Rice University. We also thank Michael Berenbrink, University of Liverpool, and Jayashree Soman, Rice University, for reading and editing both the original and revised manuscripts.

      Author Profile

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