1995
DOI: 10.1021/bi00046a002
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Abstract: A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 ox… Show more

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Cited by 118 publications
(125 citation statements)
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References 46 publications
(67 reference statements)
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“…11), we have now used site-directed disulfide crosslinking of coexpressed permease fragments, each with a single Cys residue at a defined position, a technique introduced to study tertiary interactions in visual rhodopsin (25). As shown, crosslinking is evident between a Cys residue at position 242 or 245 (helix VII) and a Cys residue at position 52 or 53 (helix II).…”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…11), we have now used site-directed disulfide crosslinking of coexpressed permease fragments, each with a single Cys residue at a defined position, a technique introduced to study tertiary interactions in visual rhodopsin (25). As shown, crosslinking is evident between a Cys residue at position 242 or 245 (helix VII) and a Cys residue at position 52 or 53 (helix II).…”
Section: Discussionmentioning
confidence: 99%
“…Yu et al (25) have described a method for determining tertiary contacts between transmembrane domains in polytopic membrane proteins based on disulfide formation between paired Cys residues in purified segments of visual rhodopsin. Because coexpression of lac permease in two fragments leads to functional complementation (26,27), we have extended this approach to show in situ that helices I and II are in close proximity to helices VII and XI in the folded tertiary structure of lac permease.…”
Section: )mentioning
confidence: 99%
“…For disulfide cross-linking, the membrane preparation (100 g of protein) in PBS (100 l) was oxidized with Cu:phen (100 M:400 M) (28,42) for 5 min at 25°C. The reaction was terminated by adding 10 mM N-ethylmaleimide and 1 mM EDTA (final concentrations).…”
Section: Methodsmentioning
confidence: 99%
“…Bovine rod transducin was purified from bovine retinas as described previously (16)(17)(18). To assay the ability of the opsin to activate transducin, we followed the increase of radioactive GTPγS bound to transducin with time after addition of retinoid in the dark or after illumination.…”
Section: Transducin Activation Assaymentioning
confidence: 99%