Determination of transmembrane protein structure by disulfide cross-linking: the Escherichia coli Tar receptor.
We have devised a generally applicable strategy for analysis of protein structure and have applied it to examine the structure of the transmembrane portion of the Tar receptor of Escherichia coli. The basis of our approach is the use of disulfide cross-linking to identify residues that are within close proximity. To generate and test large numbers of cysteine pairs, we used an unusual method of mutagenesis by which cysteine substitutions can be created randomly at a number of targeted codons. Cysteine-substituted proteins encoded by mutagenized genes may be screened directly for disulfide formation within oligomers or, alternatively, different pools of genes may be randomly recombined to generate gene populations with substitutions in multiple regions. Thus, it is possible to detect a variety of disulfide cross-links between and within individual protein molecules. Interactions between the four membrane-spanning stretches of the Tar dimer were probed by measuring the tendency of 48 cysteine substitutions throughout this region to form disulfide cross-links with one another. We have interpreted these data to suggest a helical-bundle structure for the transmembrane region. The four helices of this bundle are not structurally equivalent: the two TM1 helices interact closely, whereas the TM2 helices are more peripherally located.
Pakula
A A
AA
Division of Biology, California Institute of Technology, Pasadena 91125.
Simon
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MI
D001426:Bacterial Proteins / Q000648:ultrastructure*; D002628:Chemoreceptor Cells; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000648:ultrastructure*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000648:ultrastructure; D015398:Signal Transduction
D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D003545:Cysteine