Antibody Imprint of a Membrane Protein Surface

Abstract

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an an-tibody " imprint " of the Cyt b surface. Amino acid se-quences were selected from a random nonapeptide phage-display library by their affinity for the mono-clonal antibody 44.1 binding site, which recognizes the native conformation of the p22 phox subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conforma-tion of a synthetic peptide derived from phage-dis-played sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29 TAGRF 33 and 183 PQVNPI 188 from discontinuous regions of p22 phox . Al-though these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the trans-membrane protein. NMR constraints, measured from the antibody-bound conformation of a composite pep-tide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing struc-tural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins. Human phagocyte flavocytochrome b (Cyt b) 1 is an electron transferase that directs metabolic electrons across the plasma