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 surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed 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 29TAGRF33 and 183PQVNPI188 from discontinuous regions of p22. Although 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 transmembrane 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 native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.
Burritt
J B
JB
Departments of Microbiology, Montana State University, Bozeman, Montana 59717, USA.
Busse
S C
SC
Gizachew
D
D
Siemsen
D W
DW
Quinn
M T
MT
Bond
C W
CW
Dratz
E A
EA
Jesaitis
A J
AJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D003573:Cytochrome b Group / Q000737:chemistry* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D000939:Epitopes; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D011994:Recombinant Proteins