Prediction of an inter-residue interaction in the chaperonin GroEL from multiple sequence alignment is confirmed by double-mutant cycle analysis

Amnon Horovitz, Elena S. Bochkareva, Ofer Yifrach, Alexander S. Girshovich

Research output: Contribution to journalEditorial

38 Scopus citations

Abstract

A search for co-ordinated amino acid changes in the hsp60 family of chaperonins suggested that cysteine residues at positions 137 and 518 in the Escherichia coli chaperonin GroEL may interact with each other. In order to determine whether this interaction indeed exists we constructed a double-mutant cycle comprising wild-type GroEL, the single mutants Cys137→Ser and Cys518→Ser and the corresponding double mutant. The effects of the two mutations on the function of GroEL, in assisting the refolding of a non-folded protein substrate (rhodanese), are shown to be non-additive. It is also shown that ADP by itself specifically destabilities the Cys518→Ser mutant GroEL particle with this effect being suppressed in the double mutant. The observed pattern of co-ordinated mutations in the hsp60 family of chaperonins is thus shown to reflect a real interaction, though most likely indirect, between Cys137 and Cys518 in GroEL. Our study demonstrates that patterns of co-ordinated mutations combined with double-mutant cycle analysis can provide structural informantion on interactions in a protein without an available three-dimensional structure at atomic resolution.

Original languageEnglish
Pages (from-to)133-138
Number of pages6
JournalJournal of Molecular Biology
Volume238
Issue number2
DOIs
StatePublished - 14 Apr 1994
Externally publishedYes

Keywords

  • Chaperones
  • Correlated mutations
  • Double-mutant cycles
  • GroEL
  • Heat shock proteins

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