Reconstitution of M13 bacteriophage coat protein. A new strategy to analyze configuration of the protein in the membrane.
The configuration of M13 bacteriophage coat protein in a model membrane was analyzed using protease digestion followed by gel permeation chromatography on Fractogel TSK in formic acid/ethanol. Important information is contained in the chromatographic patterns of the membrane-bound fragments, as well as of the fragments released by the digestion. A new reconstitution was thereby developed which involves adding a small volume of a concentrated solution of cholate-solubilized coat protein to preformed vesicles (with the amount of detergent added being less than that required to solubilize the vesicles), freezing in liquid nitrogen, thawing, followed by dialysis to remove excess detergent. The coat protein is incorporated with high efficiency (95 percent) making subsequent fractionation unnecessary. In addition, the incorporated protein is not aggregated, and is incorporated with most molecules spanning the membrane, oriented in the same manner as in vivo (N-terminus outwards). Two previously described reconstitutions, using sonication or cholate dialysis, are analyzed and found to be less satisfactory.
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003090:Coliphages / Q000378:metabolism*; D019286:Endopeptidase K; D010450:Endopeptidases; D004926:Escherichia coli / Q000378:metabolism; D008051:Lipid Bilayers; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014759:Viral Envelope Proteins / Q000302:isolation & purification / Q000378:metabolism*
D008051:Lipid Bilayers; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; D010450:Endopeptidases; D019286:Endopeptidase K