A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL

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A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL. / Cliff, M J; Kad, N M; Hay, N; Lund, P A; Webb, M R; Burston, S G; Clarke, A R.

In: Journal of Molecular Biology, Vol. 293, No. 3, 29.10.1999, p. 667-84.

Research output: Contribution to journalArticlepeer-review

Harvard

Cliff, MJ, Kad, NM, Hay, N, Lund, PA, Webb, MR, Burston, SG & Clarke, AR 1999, 'A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL', Journal of Molecular Biology, vol. 293, no. 3, pp. 667-84. https://doi.org/10.1006/jmbi.1999.3138

APA

Cliff, M. J., Kad, N. M., Hay, N., Lund, P. A., Webb, M. R., Burston, S. G., & Clarke, A. R. (1999). A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL. Journal of Molecular Biology, 293(3), 667-84. https://doi.org/10.1006/jmbi.1999.3138

Vancouver

Author

Cliff, M J ; Kad, N M ; Hay, N ; Lund, P A ; Webb, M R ; Burston, S G ; Clarke, A R. / A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL. In: Journal of Molecular Biology. 1999 ; Vol. 293, No. 3. pp. 667-84.

Bibtex

@article{e007c76e1b1d4f0f84151de32e994367,
title = "A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL",
abstract = "Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.",
keywords = "Tryptophan, Fluorescence, Thermodynamics, Tyrosine, Hydrolysis, Nucleotides, Adenosine Diphosphate, Phosphates, Chaperonin 60, Kinetics, Binding, Competitive, Escherichia coli, Fluorometry, Allosteric Regulation, Models, Chemical, Adenosine Triphosphate, Amino Acid Substitution, Protein Conformation",
author = "Cliff, {M J} and Kad, {N M} and N Hay and Lund, {P A} and Webb, {M R} and Burston, {S G} and Clarke, {A R}",
note = "Copyright 1999 Academic Press.",
year = "1999",
month = oct,
day = "29",
doi = "10.1006/jmbi.1999.3138",
language = "English",
volume = "293",
pages = "667--84",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Elsevier",
number = "3",

}

RIS

TY - JOUR

T1 - A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL

AU - Cliff, M J

AU - Kad, N M

AU - Hay, N

AU - Lund, P A

AU - Webb, M R

AU - Burston, S G

AU - Clarke, A R

N1 - Copyright 1999 Academic Press.

PY - 1999/10/29

Y1 - 1999/10/29

N2 - Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.

AB - Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.

KW - Tryptophan

KW - Fluorescence

KW - Thermodynamics

KW - Tyrosine

KW - Hydrolysis

KW - Nucleotides

KW - Adenosine Diphosphate

KW - Phosphates

KW - Chaperonin 60

KW - Kinetics

KW - Binding, Competitive

KW - Escherichia coli

KW - Fluorometry

KW - Allosteric Regulation

KW - Models, Chemical

KW - Adenosine Triphosphate

KW - Amino Acid Substitution

KW - Protein Conformation

U2 - 10.1006/jmbi.1999.3138

DO - 10.1006/jmbi.1999.3138

M3 - Article

C2 - 10543958

VL - 293

SP - 667

EP - 684

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -