Design of a functional membrane protein by engineering a heme-binding site in glycophorin A.
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.
Cordova
Jeanine M
JM
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Noack
Pamela L
PL
Hilcove
Simon A
SA
Lear
James D
JD
Ghirlanda
Giovanna
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006021:Glycophorins / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D013057:Spectrum Analysis; D013997:Time Factors; D014461:Ultracentrifugation
D006021:Glycophorins; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D006418:Heme; D006639:Histidine