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!