Disulfide mutants of the binding domain of the rat low affinity nerve growth factor receptor (p75NGFR).
The binding domain of the low affinity nerve growth factor receptor (p75NGFR) is built from four "cysteine repeats," with almost identical patterns of half-cystine residues. To study the pattern of disulfide bridging within each cysteine repeat, we have mutated pairs of cysteine residues, primarily in the fourth repeat, and have tested the relative ability of each mutant to be cross-linked to the nerve growth factor after transient expression in COS 7 cells. The results give an indication of the relative importance of different disulfide bonds for the stability of the active conformation. Immunofluorescent staining shows that all of these proteins, even those that may be partially misfolded, are expressed to varying degrees on the surface of COS cells. They can also be detected by the monoclonal antibody MC192 on a Western blot following electrophoresis on a nondenaturing gel. Some cannot, however, be detected by MC192 on a Western blot after heat denaturation in SDS. The ability to refold under these conditions correlates in general with the ability of the mutant protein to bind nerve growth factor on the surface of COS cells.
Baldwin
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Department of Neurobiology, Stanford University School of Medicine, California 94305.
Shooter
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006860:Hydrogen Bonding; D008961:Models, Structural; D016296:Mutagenesis; D009414:Nerve Growth Factors / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D051381:Rats; D017475:Receptors, Nerve Growth Factor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D004220:Disulfides; D009414:Nerve Growth Factors; D017475:Receptors, Nerve Growth Factor; D003545:Cysteine