Complete assignment of neurophysin disulfides indicates pairing in two separate domains.
The pairing of the 14 half-cystine residues of bovine neurophysin was established by sequential proteolytic digestion. Purified released peptides and the residual disulfide-linked core were monitored at each step by use of amino acid analysis, gas-phase sequencing, and mass spectrometry. The approach included application of gas-phase sequencing to assign disulfide pairs in peptides containing multiple disulfides. The results demonstrate that neurophysin disulfides are paired in two distinct domains--an NH2 domain (residues 10-54) containing four disulfides and a COOH domain (residues 61-85) containing three disulfides. The specific disulfide bridges are Cys-10 to Cys-54, Cys-13 to Cys-27, Cys-21 to Cys-44, Cys-28 to Cys-34, Cys-61 to Cys-73, Cys-74 to Cys-79, and Cys-67 to Cys-85. The results place the internally duplicated segments of neurophysin (residues 12-31 and 60-77) in separate domains. Disulfide-pairing patterns within each domain are homologous with the exception of the Cys-10 to Cys-54 bond, which is unique to the NH2 domain and which links the two ends of this domain together. The potential role of the Cys-10 to Cys-54 bond in organizing the hormone-binding site is discussed.
Burman
S
S
Department of Biochemistry, Cornell University Medical College, New York, NY 10021.
Wellner
D
D
Chait
B
B
Chaudhary
T
T
Breslow
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003553:Cystine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009481:Neurophysins / Q000032:analysis* / Q000378:metabolism; D010449:Peptide Mapping; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D009481:Neurophysins; D003553:Cystine; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus