TY - JOUR
T1 - Design of a functional membrane protein by engineering a heme-binding site in glycophorin A
AU - Cordova, Jeanine M.
AU - Noack, Pamela L.
AU - Hilcove, Simon A.
AU - Lear, James D.
AU - Ghirlanda, Giovanna
PY - 2007/1/24
Y1 - 2007/1/24
N2 - 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.
AB - 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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U2 - 10.1021/ja057495i
DO - 10.1021/ja057495i
M3 - Article
C2 - 17227013
AN - SCOPUS:33846436807
SN - 0002-7863
VL - 129
SP - 512
EP - 518
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 3
ER -