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Multiplexed Electrochemistry of DNA-Bound Metalloproteins

Pheeney, Catrina G. and Arnold, Anna R. and Grodick, Michael A. and Barton, Jacqueline K. (2013) Multiplexed Electrochemistry of DNA-Bound Metalloproteins. Journal of the American Chemical Society, 135 (32). pp. 11869-11878. ISSN 0002-7863. PMCID PMC3899834. http://resolver.caltech.edu/CaltechAUTHORS:20130913-110347612

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Abstract

Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of endonuclease III (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/ja4041779 DOIArticle
http://pubs.acs.org/doi/abs/10.1021/ja4041779PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899834/PubMed CentralArticle
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2013 American Chemical Society. Received: April 26, 2013; Published: July 30, 2013. We are grateful for the financial support of the National Institutes of Health (GM49216) and ONR (N00014-09-1-1117). A.R.A. was supported by the National Institute on Aging of the NIH on a predoctoral NRSA (F31AG040954). The authors also thank the Kavli Nanoscience Institute facilities and staff for help in streamlining the fabrication of multiplexed chips.
Group:Kavli Nanoscience Institute
Funders:
Funding AgencyGrant Number
NIHGM49216
Office of Naval Research (ONR)N00014-09-1-1117
NIH Predoctoral FellowshipF31AG040954
National Institute on AgingUNSPECIFIED
PubMed Central ID:PMC3899834
Record Number:CaltechAUTHORS:20130913-110347612
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20130913-110347612
Official Citation:Multiplexed Electrochemistry of DNA-Bound Metalloproteins Catrina G. Pheeney, Anna R. Arnold, Michael A. Grodick, and Jacqueline K. Barton Journal of the American Chemical Society 2013 135 (32), 11869-11878
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41321
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:16 Sep 2013 21:10
Last Modified:21 Jul 2017 22:51

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