Membrane protein topology determination by proteolysis of maltose binding protein fusions.
A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest. Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain. A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm. A cytosolic MBP domain (MBP delta 2-26) is attached to the protein when its N-terminus resides in the cytoplasm. The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop. Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP. The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments. Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain. The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments. Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them.
Miller
K W
KW
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Konen
P L
PL
Olson
J
J
Ratanavanich
K M
KM
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C461599:maltose transport system, E coli; C034811:tetracycline resistance-encoding transposon repressor protein; D010447:Peptide Hydrolases