NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution

被引:46
作者
Kobayashi, N
Freund, SMV
Chatellier, J
Zahn, R
Fersht, AR
机构
[1] Univ Cambridge, Chem Lab, Cambridge CB2 2QH, England
[2] MRC, Ctr Prot Engn, Cambridge CB2 2QH, England
关键词
Hsp60; maltose-binding protein; protein-peptide interactions; NMR; transferred NOE;
D O I
10.1006/jmbi.1999.3042
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an a-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins. (C) 1999 Academic Press.
引用
收藏
页码:181 / 190
页数:10
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