Journal
BIOCHEMICAL SOCIETY TRANSACTIONS
Volume 41, Issue -, Pages 35-42Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BST20120212
Keywords
cysteine accessibility; dual topology reporter; N-ethyl-maleimide; tag insertion
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Funding
- Swiss National Science Foundation [31-67188.01, CRSI33_125232/1]
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Most integral membrane proteins of yeast with two or more membrane-spanning sequences have not yet been crystallized and for many of them the side on which the active sites or ligand-binding domains reside is unknown. Also, bioinformatic topology predictions are not yet fully reliable. However, so-called low-resolution biochemical methods can be used to locate hydrophilic loops or individual residues of polytopic membrane proteins at one or the other side of the membrane. The advantages and limitations of several such methods for topological studies with yeast ER integral membrane proteins are discussed. We also describe new tools that allow us to better control and validate results obtained with SCAM (substituted cysteine accessibility method), an approach that determines the position of individual residues with respect to the membrane plane, whereby only minimal changes in the primary sequence have to be introduced into the protein of interest.
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