Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.
We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed.
Newton
S M
SM
Unité de Programmation Moléculaire et Toxicologie Génétique, Centre National de la Recherche Scientifique Unité de Recherche Associée 1444, Institut Pasteur, Paris, France.
Klebba
P E
PE
Michel
V
V
Hofnung
M
M
Charbit
A
A
D001425:Bacterial Outer Membrane Proteins; D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D000939:Epitopes; D008565:Membrane Proteins / Q000276:immunology / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018272:Porins / Q000276:immunology / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000276:immunology / Q000648:ultrastructure*; D013329:Structure-Activity Relationship; D013499:Surface Properties; D014961:X-Ray Diffraction
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D000939:Epitopes; D008565:Membrane Proteins; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins