ANTIBODY IMPRINT OF A MEMBRANE-PROTEIN SURFACE - PHAGOCYTE FLAVOCYTOCHROME-B

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody ''imprint'' of the Cyt b su rface. Amino acid sequences were selected from a random nonapeptide ph age-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22(pho x) subunit of Cyt b, Transferred nuclear Overhauser effect spectroscop y and rotating frame Overhauser effect spectroscopy NMR were used to s tudy the antibody-bound conformation of a synthetic peptide derived fr om phage-displayed sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed th e modeling of the close spatial proximity of the epitope components (2 9)TAGRF(33) and (183)PQVNPI(188) from discontinuous regions of p22(pho x). Although these regions are separated by two putative membrane-span ning domains and are 150 residues apart in the sequence, they appear t o combine to form a complex epitope on the cytosolic surface of the tr ansmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide 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 nativ e discontinuous epitope on the Cyt b surface. Given additional antibod ies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low a bundance or noncrystallizable proteins.