Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants.
Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110-Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.
Hwa
J
J
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Klein-Seetharaman
J
J
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012174:Retinitis Pigmentosa / Q000235:genetics*; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D004220:Disulfides; D008301:Maleimides; D017299:Rod Opsins; C425927:maleimido-butyryl-biocytin; D012243:Rhodopsin; D019286:Endopeptidase K; D008239:Lysine; D003545:Cysteine