Boris M. Gorovits

Productive and Nonproductive Intermediates in the Folding of Denatured Rhodanese

The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 m urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species.

The chaperonin GroEL is destabilized by binding of ADP.

The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4′-bis(1-anilino-8-naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at 1 M urea. The same destabilization by ADP and stabilization by Mg were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5′-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed.

Conditions for Nucleotide-dependent GroES-GroEL Interactions GroEL14(GroES7)2 IS FAVORED BY AN ASYMMETRIC DISTRIBUTION OF NUCLEOTIDES

A still unresolved question regarding the mechanism of chaperonin-assisted protein folding involves the stoichiometry of the GroEL-GroES complex. This is important, because the activities of the Escherichia coli chaperonin GroEL are modulated by the cochaperonin GroES. In this report, the binding of GroES to highly purified GroEL in the presence of ATP, ADP, and the nonhydrolyzable ATP analogue, 5′-adenylyl β,γ-imidodiphosphate (AMP-PNP), was investigated by using the fluorescence anisotropy of succinimidyl-1-pyrenebutyrate-labeled GroES. In the presence of Mg2+-ATP and high [KCl] (10 mm), two GroES7 rings bind per one GroEL14. In contrast, in the presence of ADP or AMP-PNP only one molecule of oligomeric GroES can be tightly bound by GroEL. With AMP-PNP, binding of a small amount (<20%) of a second GroES can be detected. In the presence of ADP alone, a second GroES ring can bind to GroEL weakly and with negative cooperativity. Strikingly, addition of AMP-PNP to the solution containing preformed GroEL14(GroES7) complexes formed in the presence of ADP results in an increase in the fluorescence anisotropy. Analysis of this effect indicates that 2 mol of GroES oligomer can be bound in the presence of mixed nucleotides. A similar conclusion follows from studies in which ADP is added to an GroEL14 (GroES7) complex formed in the presence of AMP-PNP. This is the first demonstration of an asymmetric distribution of nucleotides bound on the 1:2 GroEL14 (GroES7)2 complex. The relation of the observed phenomena to the proposed mechanism of the GroEL function is discussed.

ATP Hydrolysis Is Critical for Induction of Conformational Changes in GroEL That Expose Hydrophobic Surfaces

The degree of hydrophobic exposure in the molecular chaperone GroEL during its cycle of ATP hydrolysis was analyzed using 1,1′-bis(4-anilino)naphthalene-5,5′disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment. In the presence of 10 mM MgCl2 and 10 mM KCl the addition of ATP, but not ADP or AMP-PNP, resulted in a time-dependent, linear increase in the bisANS fluorescence. The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe. The effect could be substantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluorescence was correlated with ATP hydrolysis. The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of bisANS. This uncompetitive inhibition suggests that the probe can interact with the GroEL-ATP complex. The inability of the nonhydrolyzable ATP analog, AMP-PNP, to cause a similar effect is explained by the interaction of bisANS with a transient conformational state of GroEL formed consequent to ATP hydrolysis. It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that results from electrostatic repulsion between the bound products of ATP hydrolysis, and it plays an important role in the mechanism of the chaperonin cycle.

FLUORESCENCE ANISOTROPY METHOD FOR INVESTIGATION OF GROEL-GROES INTERACTION

Publisher Summary The chapter presents a study related to GroEL-GroES interaction through fluorescence anisotropy method. The molecular chaperones GroEL and GroES have been shown to assist proteins in their folding and assembly in vivo and in vitro. The ability to investigate stoichiometry of the GroEL 14 -GroES 7 interaction in solution under physiological conditions is important. The chapter describes a direct method of detecting GroEL-GroES interaction in solution by using fluorescently labeled co-chaperonin GroES and applying a fluorescence anisotropy assay method. Fluorescence anisotropy measurements have been widely used to detect change in the rotational diffusion properties of many proteins. This method has been applied to quantify protein-ligand and protein-protein association reactions. The investigated protein is initially labeled with a fluorophore with appropriate fluorescence lifetime. The sample is then excited with vertically polarized light. To estimate the stoichiometry of the GroEL-GroES interaction under different conditions of binding, pyrene-labeled GroES 7 oligomer is titrated to the solution containing a fixed known amount of GroEL 14 . The chapter describes the methods of GroES labeling with succimidyl-l-pyrene butyrate and GroES binding to GroEL.