A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL.
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.
Cliff
M J
MJ
Department of Biochemistry School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK. M.J.Cliff@Bris.ac.uk
Kad
N M
NM
Hay
N
N
Lund
P A
PA
Webb
M R
MR
Burston
S G
SG
Clarke
A R
AR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D005453:Fluorescence; D005470:Fluorometry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D018834:Chaperonin 60; D009711:Nucleotides; D010710:Phosphates; D014443:Tyrosine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate