Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study.
The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.
Sot
Begoña
B
Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, 48080 Bilbao, Spain.
von Germar
Fritzthof
F
Mäntele
Werner
W
Valpuesta
Jose María
JM
Taneva
Stefka G
SG
Muga
Arturo
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D001665:Binding Sites; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D017354:Point Mutation; D011487:Protein Conformation; D015398:Signal Transduction; D013055:Spectrophotometry, Infrared; D055672:Static Electricity
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D007477:Ions; D000255:Adenosine Triphosphate