title: Determining the topology of an integral membrane protein.
abstract: A variety of methods have been developed for assigning the aqueous domains of integral membrane proteins to either side of a biological membrane. Once the sequence of a protein is known from its DNA sequence it is possible to study the topology of the protein. This unit provides protocols in which the water-soluble domains can be tested for their accessibility to reagents added to membranes with a defined orientation. Tagging of hydrophilic regions of the protein with different epitopes and probing of their orientation with respect to the membrane is also described. Finally, a procedure for fusion of a reporter enzyme to truncated fragments of the protein is provided. The fusion protein is used as a sensor of sequence disposition relative to the membrane.
authors.affiliation: Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: N
authors.initials: N
authors.lastname: Green
authors.forename: H
authors.initials: H
authors.lastname: Fang
authors.forename: K U
authors.initials: KU
authors.lastname: Kalies
authors.forename: V
authors.initials: V
authors.lastname: Canfield
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000276:immunology / Q000648:ultrastructure*; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000648:ultrastructure; D012995:Solubility; D014867:Water
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014867:Water; D010447:Peptide Hydrolases