Protein Engineering Aequorin Mutagenesis
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Transcript of Protein Engineering Aequorin Mutagenesis
E.DIKICI ET AL (2009) .
Aequorin variants with improved bioluminescence
properties
António Sousa64427 MBioNano
Luminescent Proteins
Introduction
■ Gene expression analysis;
■ Drug discovery;
■ Study of protein dynamics;
■ Mapping signal transduction pathways.
Produce variants of the bioluminescent photoprotein, aequorin, with different bioluminescence lifetimes and/or emission wavelengths.
Bioluminescent Proteins
Goal:
■ Low background signal;
■ High sensitivity for biological sample analysis.
Alternative!
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)
Is stabilized by H-bonds,π–π interactions and hydrophobic interactions within the active site.
Native coelenterazine
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.
Methods
Rational site-directed mutagenesis.
Mismatches in base pairing
Mismatches in base pairing
DNA Polymerase
dNTPs
■ 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.
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
Methods
1 - Expression and purification of mutant apoaequorin variants from E.coli
The coding sequence for aequorin was ligated into a vector containing the lpp promoter and ompA leader sequence.
Culture of cells
Extraction of proteins
Purification of proteins
Lyophilization
Apoaequorin was 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!
The Study
Hydrogen Bonding and
π-π interactions
The X-ray crystal structure of aequorin reveals a 600 A° hydrophobic core in which coelenterazine resides. – 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 coelenterazine i (left), and coelenterazine hcp.
Bioluminescence
Use of different analogues of the chromophore with apoaequorin to explore the effect of different coelenterazine structures on the bioluminescence of the protein.
Cystein-free aequorin with native coelenterazine
Aequorin mutant w86f with colentererazine hcp.
Aequorin mutant Y82F with colentererazine i.
By decreasing the size of the hydrophobic side chain of the colentererazine, the effectiveness of hydrophobic packing isreduced.
Shift in emission maxima.
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!
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!