Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis.
We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].
Young
Y
Y
Department of Protein Structure, Amgen, Inc, Thousand Oaks, CA 91320, USA.
Zeni
L
L
Rosenfeld
R D
RD
Stark
K L
KL
Rohde
M F
MF
Haniu
M
M
D054369:Agouti-Related Protein; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C515198:AGRP protein, human; D054369:Agouti-Related Protein; D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; D003545:Cysteine; D005033:Ethylmaleimide