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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

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DNA targeting and cleavage by an engineered metalloprotein dimer.

Siu Wah Wong-Deyrup¹, Charulata Prasannan, Cynthia M Dupureur

¹Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.

Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry

|November 26, 2011

Summary

Engineered protein dimers show enhanced calcium binding and DNA cleavage activity. This advancement in artificial metallonuclease design utilizes biologically relevant metal ions for unique functions.

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Area of Science:

Protein Engineering
Biochemistry
Molecular Biology

Background:

Protein dimerization offers structural and functional benefits.
A previously designed metallohomeodomain protein (C2) binds metal ions and DNA with limited activity.
EF-hand and homeodomain motifs were combined to create the C2 protein.

Purpose of the Study:

To enhance the function of the engineered metalloprotein C2.
To create a dimeric form of the C2 protein (F2) for improved performance.
To investigate the DNA cleavage activity of the dimeric metalloprotein.

Main Methods:

Fluorescence spectroscopy was used to assay calcium binding.
Gel shift selection experiments determined DNA sequence specificity.
Plasmid DNA cleavage assays were performed.

Main Results:

The dimeric F2 protein exhibited 25-fold higher calcium (Ca(II)) binding affinity per domain compared to the C2 monomer.
Metallated F2 showed specificity for 5'-TAATTA-3' DNA sequences.
The Ca(2)F2 complex demonstrated calcium-dependent plasmid DNA cleavage, unlike the monomer.

Conclusions:

Dimerization significantly enhances the functional properties of the engineered metalloprotein.
The dimeric metalloprotein F2 represents a novel artificial metallonuclease with Ca(II)-dependent DNA cleavage.
This work advances the design of artificial metallonucleases using biologically significant metal ions.