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Home / Equine Research Bank / Biochim Biophys Acta / A myoglobin variant with a polar substitution in a conserved...
Biochimica et biophysica acta1997; 1341(1); 1-13; doi: 10.1016/s0167-4838(97)00064-2

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Abstract: Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.
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Publication Date: 1997-08-15 PubMed ID: 9300804DOI: 10.1016/s0167-4838(97)00064-2Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research examines the effect of a polar amino acid substitution in a conserved hydrophobic cluster of the heme binding pocket of horse heart myoglobin, specifically investigating the impact on protein stability. The study reveals that despite the lack of significant structural differences between the mutant and wild-type myoglobins, the introduced change reduces the thermal stability of the protein.

Study Design and Methodology

  • The researchers synthetically modified a horse heart myoglobin protein, replacing the Leucine (Leu) amino acid at the position 104 with an Asparagine (Asn), a polar amino acid of a similar size. This variant protein is termed Leu104Asn mutant.
  • This mutant protein, as well as its recombinant wild-type counterpart, were expressed from synthetic genes in Escherichia coli, a common host for gene expression in laboratory settings.
  • Thermal denaturation (unfolding of the proteins due to heat) of the two myoglobins was studied by measuring the change in circular dichroism ellipticity at 222 nanometers (a specific wavelength in the UV spectrum), which is an effective way to monitor protein secondary structure changes.

Results

  • Analysis of thermal denaturation revealed the Leu104Asn mutant had significantly lower thermal stability when compared to the recombinant wild-type myoglobin.
  • Structural examination using X-ray diffraction techniques revealed no significant structural differences between the mutant and wild-type myoglobins despite the lower thermal stability of the Leu104Asn mutant.
  • EPR (Electron Paramagnetic Resonance) and absorption spectroscopic analyses confirmed the similarity between the heme iron centres in both proteins, indicating that the mutation did not significantly affect the structure of the protein.

Conclusions

  • The observed decrease in thermal stability of the mutant protein despite no identifiable structural differences implies that the introduction of a polar asparagine into a hydrophobic environment perturbs important helix-helix and heme-protein interactions.
  • The research suggests that well-ordered internal amino acids contribute significantly to protein stability and that perturbations can induce structural destabilization, reflecting in a significant decrease in the thermal stability of the myoglobin.

Cite This Article

APA
Maurus R, Overall CM, Bogumil R, Luo Y, Mauk AG, Smith M, Brayer GD. (1997). A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket. Biochim Biophys Acta, 1341(1), 1-13. https://doi.org/10.1016/s0167-4838(97)00064-2

Publication

Biochimica et biophysica acta
ISSN: 0006-3002
NlmUniqueID: 0217513
Country: Netherlands
Language: English
Volume: 1341
Issue: 1
Pages: 1-13

Researcher Affiliations

Maurus, R
  • Department of Biochemistry and Molecular Biology, and the Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Overall, C M
    Bogumil, R
      Luo, Y
        Mauk, A G
          Smith, M
            Brayer, G D

              MeSH Terms

              • Animals
              • Asparagine / chemistry
              • Binding Sites
              • Circular Dichroism
              • Electron Spin Resonance Spectroscopy
              • Escherichia coli / genetics
              • Heme / metabolism
              • Horses
              • Leucine / chemistry
              • Molecular Sequence Data
              • Mutation
              • Myocardium / metabolism
              • Myoglobin / chemistry
              • Myoglobin / genetics
              • Myoglobin / metabolism
              • Nucleic Acid Conformation
              • Protein Conformation
              • Protein Structure, Secondary
              • Recombinant Proteins / chemistry
              • Recombinant Proteins / genetics
              • Spectrophotometry

              Citations

              This article has been cited 32 times.
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