Web Release Date: December 29,
Design of a Functional Membrane Protein by Engineering a Heme-Binding Site in Glycophorin A
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Contribution from the Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received November 2, 2005
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.
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