Trp203 mutation in GroEL promotes a self-association reaction: a hydrodynamic study.

A combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge is used to investigate the hydrodynamic integrity and increased self-association interactions of the mutant GroEL Y203W when compared to the wild-type GroEL molecule, which may be derived from increased hydrophobic exposure caused by the mutation. Sedimentation velocity has revealed that three distinct species were present throughout the concentration ranges used, corresponding to 14-mer (GroEL "super monomer") and 28-mer ("super dimer") subunit compositions with a small amount of 42-mer ("super trimer"), which, from the relative concentration of each species, would give an estimated weight average molecular weight of (1.0 +/- 0.1) x 10(6) Da. Sedimentation equilibrium gave an apparent weight average molecular weight (Mw,app) of (910,000 +/- 5000) Da, which is in agreement with these findings. These results are in contrast to wild-type GroEL which, in excellent agreement with the previous findings of Behlke and co-workers, revealed a single species with an Mw,app of (805,000 +/- 5200) Da and a sedimentation coefficient s(0)20,w of (21.6 +/- 0.3) S. We therefore conclude that the tryptophan mutation at the Y203 location causes a significant degree of self-association of the GroEL 14-mer assembly (with dimer and trimer present). These findings would appear to correlate well with the findings of Gibbons et al., who showed an increase in hydrophobic exposure due to this mutation.

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.

Scaleup of a pharmaceutical granulation in planetary mixers.

The scaleup of a pharmaceutical granulation in a series of planetary mixers with increasing bowl capacity between 5 and 200 liters has been studied by applying the classical dimensionless numbers of Power, Reynolds, and Froude to define the power consumption profile of each mixer as a function of the processing conditions in dimensionless form. Experiments were performed using a formulation based on dicalcium phosphate dihydrate containing pregelatinized starch, with water being added at a fixed rate. Samples were taken at different power consumptions and assessed using mixer torque rheometry. Geometrically similar machines gave the same dimensionless relationship, but when similarity was compromised by the use of modified bowls and blades, different relationships occurred. The results show that, for geometrically similar machines, it is possible to calculate the power consumption at a predefined granulation endpoint at any given operating condition at any scale.

Applicability of a scale-up methodology for wet granulation processes in Collette Gral high shear mixer-granulators.

This study investigates the extension of a scale-up methodology based on dimensionless power relationships, to a series of vertical mixer-granulators in which the bowl is removable, i.e. both impeller and chopper blades are mounted on top-driven vertical shafts positioned through the lid. Granulation runs were carried out in several bowl sizes belonging to the series of Collette Gral mixer-granulators, ranging from 8 to 600 l capacities. It was shown that under certain conditions a common scale-up master curve could be drawn from the data gathered for each bowl, thus permitting the use of such a curve for the determination of mixer-granulator power consumption at a defined granulation end-point. The results also helped to clarify the concept of similitude, both geometrical and dynamic, which is implied in the methodology. The importance of wall slippage (as promoted by the insertion of a PTFE lining into the bowls) and batch size are illustrated.

Process control in a high shear mixer-granulator using wet mass consistency: the effect of formulation variables.

This work investigates the relationships between the wet mass consistency/viscosity of samples prepared in a mixer-granulator and physical properties of the dry granules produced from the wet mass; namely, size distribution, bulk density (Hausner ratio), friability, and flow avalanching behavior. The correlation between the consistency of the wet mass and the downstream dry granule properties confirms that consistency is the key parameter to control in wet granulation by mechanical agitation. Variations in the formulation affect the dimensionless power relationship of the mixer-granulator considered; that is, the equivalence between wet mass consistency and mixer net power consumption, which is actually the parameter used to monitor the wet granulation process. The same variations in formulation also affect the relationships between wet mass consistency and dry granule properties.

A methodology for the optimization of wet granulation in a model planetary mixer.

This paper investigates a methodology for the optimization of wet granulation processes in planetary mixers. A model formulation was granulated in a planetary mixer (two different bowl sizes). The wet masses were characterized by their bulk density and consistency (as measured by mixer torque rheometry), and the feasibility of scale-up from one mixer bowl to the other was studied using a dimensionless numbers approach for the estimation of the power consumption at the granulation end point. Both bowls gave the same dimensionless power relationships (a relationship between the power number, Reynolds number, Froude number, and bowl fill ratio), which could therefore be used for calculating the power consumption level when the wet mass achieves its target values of density and consistency, i.e., the point at which granulation should be stopped. It was also shown that batches granulated in different conditions (batch size, blade speed) in two planetary mixers, but presenting similar wet mass characteristics (bulk density and consistency) led to dry granules of similar properties: granule size distribution, density, friability, and flow. This work suggests that it is possible to characterize the wet mass by only two parameters which describe the quality of the downstream granules. The scale-up procedure based on the use of dimensionless numbers was found to be applicable to planetary mixers, provided they give one common dimensionless power relationship.

Asymmetry, commitment and inhibition in the GroE ATPase cycle impose alternating functions on the two GroEL rings.

The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides. The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state. However, ADP binding completely inhibits ATP hydrolysis in the asymmetric complex, i.e. ATP cannot by hydrolysed when ADP is bound to the other ring. This non-competitive inhibition of the ATPase by ADP is consistent with a ring-switching, or "two-stroke", mechanism of the type: ATP:GroEL --> ADP:GroEL --> ADP:GroEL:ATP --> GroEL:ATP --> GroEL:ADP, i.e. with respect to the GroEL rings, ATP turns over in an alternating fashion. When the ATP-stabilized, "open" state is challenged with hexokinase and glucose, to quench the free ATP, the open state relaxes slowly (0.44 s-1) back to the apo (or closed) conformation. This rate, however, is three times faster than the hydrolytic step, showing that bound ATP is not committed to hydrolysis. When GroES is bound to the GroEL:ATP complex and the system is quenched in the same way, approximately half of the bound ATP undergoes hydrolysis on the chaperonin complex showing that the co-protein increases the degree of commitment. Thus, non-competitive inhibition of ATP hydrolysis, combined with the ability of the co-protein to block ligand exchange between rings has the effect of imposing a reciprocating cycle of reactions with ATP hydrolysing, and GroES binding, on each of the GroEL rings in turn. Taken together, these data imply that the dominant, productive steady state reaction in vivo is: GroEL:ATP:GroES --> GroEL:ADP:GroES --> ATP:GroEL:ADP:GroES --> ATP:GroEL:ADP --> GroES:ATP:GroEL:ADP --> GroES:ATP:GroEL for a hemi-cycle, and that significant inhibi tion of hydrolysis may arise through the formation of a dead-end ADP:GroEL:ATP:GroES complex.

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels.

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue explants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I- or D-asparate from mammary tissue explants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.