Determining the topology of an integral membrane protein.
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.
Green
N
N
Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Fang
H
H
Kalies
K U
KU
Canfield
V
V
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
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014867:Water; D010447:Peptide Hydrolases