Protein Engineering Aequorin Mutagenesis

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  • 1. Aequorin variants with improved bioluminescence properties
    E.Dikici et al (2009).
    António Sousa
    64427 MBioNano
  • 2. Luminescent Proteins
    Introduction
    Alternative!
    ■ Gene expression analysis;
    ■ Drug discovery;
    ■ Study of protein dynamics;
    ■ Mapping signal transduction pathways.
    ■ Low background signal;
    ■ High sensitivity for biological sample analysis.
    Produce variants of the bioluminescent photoprotein, aequorin, with different bioluminescence lifetimes and/or emission wavelengths.
    Bioluminescent Proteins
    Goal:
  • 3. Introduction
    Native aequorin:
    + Apoaequorin (189 amino acid residues);
    + Molecular Oxygen;
    3 binding sites for calcium.
    The protein undergoes a conformational change that triggers the oxidation of the chromophore, resulting in the emission of light (λ=469 nm)
    Native coelenterazine
    Is stabilized by H-bonds,π–π interactions and hydrophobic interactions within the active site.
  • 4. Introduction
    Rational site-directed mutagenesis.
    Incorporation with coelenterazine analogues.
    Two expression Systems. Bacillus subtilis and Escherichia coli.
    Expression plasmids containing the gene of the cysteine-free mutant of apoaequorin were used as a template for the PCR.
    Site-directed mutagenesis was confirmed through DNA sequencing.
  • 5. Methods
    Rational site-directed mutagenesis.
    Mismatches in base pairing
    ■ A primer that contains a few mismatches can steel anneal to its target DNA so as to permit initiation of DNA synthesis
    ■ The amplified product contains fragments that are exactly complementary to the mismatched primer (witch is no longer mismatched).
    ■ This is a convenient method to engineer sequence changes into a target DNA.
    Mismatches in base pairing
    DNA Polymerase
    dNTPs
  • 6. Methods
    Plasmid DNA expressed in E.coli XL-1 Blue.
    Transformation of E.coli JM109. (1)
    Plasmid DNA expressed in E.coli JM109.
    Transformation of B.subtilis cells. (2)
    2 - Expression and purification of mutant apoaequorin variants from subtilis
    Culture of cells
    Extraction of proteins
    Purification of proteins
    Lyophilization
  • 7. Methods
    1 - Expression and purification of mutant apoaequorin variants from E.coli
    The coding sequence for aequorin was ligated into a vector containing the lpppromoter and ompA leader sequence.
    Culture of cells
    Extraction of proteins
    Purification of proteins
    Lyophilization
    Apoaequorinwas usually ≥95% pure
    Generation of the mutant aequorin variants from their respective apoaequorins was achieved by mixing purified protein with a two to three molar excess of a coelenterazine analogue.
    Study of bioluminescence activity, half-life and stability!
  • 8. The Study
    The X-ray crystal structure of aequorin reveals a 600 A° hydrophobic core in which coelenterazineresides. – 21 residues that stabilize chromophore.
    Mutating residues, His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132.
    Any mutations involving these residues can result in emission shifts, as long as the changes do not destabilize the coelenterazine molecule to such extent that the activity is lost.
    Chemical structures of coelenterazinei (left), and coelenterazinehcp.
  • 9. Bioluminescence
    By decreasing the size of the hydrophobic side chain of the colentererazine, the effectiveness of hydrophobic packing is
    reduced.
    Use of different analogues of the chromophore with apoaequorin to explore the effect of different coelenterazine structures on the bioluminescence of the protein.
    Shift in emission maxima.
    Cystein-free aequorin with native coelenterazine
    Aequorin mutant w86f with colentererazine hcp.
    Aequorin mutant Y82F with colentererazine i.
  • 10. Half-Life
    Therefore, when combining time-resolved approach with the spatial resolution, it is possible to detect up to four different analytes within a given sample.
    Multi-analyte detection tool!
  • 11. Long Term Stability
    These results suggest that the mutations made within the active site of the aequorin can be detrimental to the long-term stability of aequorin!