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 transiti ons spectrofluorometrically. The tyrosine residues at positions 203, 360, 4 76 and 485 were mutated. Of these, the probe at residue 485 gave the deares t fluorescence signals upon nucleotide binding. The probe at 360 reported s imilar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated ti mescales, all of which precede hydrolysis of the nucleotide. All four of th ese rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is prop osed to be a T-to-R transition. The next kinetic phase is a rearrangement t o the open state identified by electron cryo-microscopy and this we designa te 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 w ith the occupied ring behaving in a concerted, cooperative manner. At highe r ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP(14) species can t hen undergo two final rearrangements, RR* --> [RR](+) --> [RR](#). These fi nal slow steps are completely blocked when ADP occupies the second ring, i. e. it does not occur in the GroEL:ATP(7):ADP(7) or the GroEL:ATP(7) 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](#), w ith concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by interring contacts. Th is kinetic behaviour is consistent with the GroEL ring passing through dist inct 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*-for m retaining high GroES affinity but allowing the substrate to dissociate in to the enclosed cavity (folding). ADP induces only one detectable rearrange ment (designated T to T*) which has no properties in common with those elic ited by ATP. However, asymmetric ADP binding prevents ATP occupying both ri ngs and, hence, restricts the system to the T*T, T*R and T*R* complexes. (C ) 1999 Academic Press.