Design of a functional membrane protein by engineering a heme-binding site in glycophorin A

Jeanine M. Cordova, Pamela L. Noack, Simon A. Hilcove, James D. Lear, Giovanna Ghirlanda

Research output: Contribution to journalArticle

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Abstract

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Out of the 32 residues comprising the transmembrane helix of GpA, five amino acids were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis, and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 ± 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Moreover, ME1 catalyzes the hydrogen peroxide dependent oxidation of organic substrates such as TMB (2,2′,5,5′-tetramethyl-benzidine). This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.

Original languageEnglish
Pages (from-to)512-518
Number of pages7
JournalJournal of the American Chemical Society
Volume129
Issue number3
DOIs
Publication statusPublished - Jan 24 2007

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Glycophorin
Protein Engineering
Binding sites
Heme
Membrane Proteins
Binding Sites
Proteins
Membranes
Oligomerization
Dimerization
Ultracentrifugation
Micelles
Electrophoresis
Histidine
Hydrogen Peroxide
Oxidation-Reduction
Spectrum Analysis
Gels
Spectroscopy
Amino Acids

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Design of a functional membrane protein by engineering a heme-binding site in glycophorin A. / Cordova, Jeanine M.; Noack, Pamela L.; Hilcove, Simon A.; Lear, James D.; Ghirlanda, Giovanna.

In: Journal of the American Chemical Society, Vol. 129, No. 3, 24.01.2007, p. 512-518.

Research output: Contribution to journalArticle

Cordova, Jeanine M. ; Noack, Pamela L. ; Hilcove, Simon A. ; Lear, James D. ; Ghirlanda, Giovanna. / Design of a functional membrane protein by engineering a heme-binding site in glycophorin A. In: Journal of the American Chemical Society. 2007 ; Vol. 129, No. 3. pp. 512-518.
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