Determining the topology of an integral membrane protein.

N. Green, H. Fang, K. U. Kalies, Victor Canfield

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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.

Original languageEnglish (US)
JournalCurrent protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
VolumeChapter 5
Publication statusPublished - May 1 2001

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All Science Journal Classification (ASJC) codes

  • Cell Biology

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