Open AccessIdentification of Amino Acid Residues at Nucleotide‐Binding Sites of Chaperonin GroEL/GroES and cpn10 by Photoaffinity Labeling with 2‐azido‐adenosine 5′‐triphosphate
Author(s) -
Bramhall Elizabeth A.,
Cross Richard L.,
Rospert Sabine,
Steede N. Kalaya,
Landry Samuel J.
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00627.x
Subject(s) - groel , groes , chaperonin , nucleotide , chemistry , photoaffinity labeling , biochemistry , stereochemistry , biology , binding site , protein folding , escherichia coli , gene
Although the chaperonin GroEL/GroES complex binds and hydrolyzes ATP, its structure is unlike other known ATPases. In order to better characterize its nucleotide binding sites, we have photolabeled the complex with the affinity analog 2‐azido‐ATP. Three residues of GroEL, Prol37, Cysl38 and Thr468, are labeled by the probe. The location of these residues in the GroEL crystal structure [Braig, K., Otwinowski, Z., Hedge, R., Boisvert, D., Joachimiak, A., Horwich, A. & Sigler, P. (1994) Nature 371 , 578–586; Boisvert, D. C., Wang, J., Otwinowski, Z., Horwich, A. L. & Sigler, P. B. (1996) Nat. Struct. Biol. 3 , 170–177] suggests that 2‐azido‐ATP binds to an alternative conformer of GroEL in the presence of GroES. The labeled site appears to be located at the GroEL/GroEL subunit interface since modification of Pro 137 and Cysl38 is most readily explained by attack of a probe molecule bound to the adjacent GroEL subunit. Labeling of the co‐chaperonin, GroES, is clearly demonstrated on gels and the covalent tethering of nucleotide allows detection of a GroES dimer in the presence of SDS. However, no stable peptide derivative of GroES could be purified for sequencing. In contrast, the GroES homolog, yeast cpnlO, does give a stable derivative. The modified amino acid is identified as the conserved Prol3, which corresponds to Pro5 in Escherichia coli GroES.