Prediction of an inter-residue interaction in the chaperonin GroEL from multiple sequence alignment is confirmed by double-mutant cycle analysis.
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 destabilizes 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 information on interactions in a protein without an available three-dimensional structure at atomic resolution.
Horovitz
A
A
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Bochkareva
E S
ES
Yifrach
O
O
Girshovich
A S
AS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D018834:Chaperonin 60; D003545:Cysteine; D004793:Enzyme Reactivators / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004793:Enzyme Reactivators; D006360:Heat-Shock Proteins; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine