Jeffrey W. Seale

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.

PHOTOINCORPORATION OF FLUORESCENT PROBE INTO GROEL : DEFINING SITE OF INTERACTION

We have elucidated conditions for the covalent incorporation of a nonspecific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in response to various effectors. This method should be useful for studying both protein structure and dynamics.

Preformed GroES oligomers are not required as functional cochaperonins.

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem.270, 30268–30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96–97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer–monomer equilibrium to be 7.3×10−34M6. The GroESD(96–97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96–97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the preformed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ-endotoxins from Bacillus thuringiensis†

The δ‐endotoxin proteins are produced by Bacillus thuringiensis during the sporulation phase of its life cycle. These proteins exhibit insecticidal activity through receptor‐mediated ion channel formation. The mode of action of these proteins requires the conversion of the protein from a water‐soluble conformation to a membrane‐inserted conformation. While there is X‐ray structure information for the soluble protein, no detailed structure exists for the membrane‐inserted protein. However, based on peptide studies, an umbrella model for the membrane‐inserted state has been proposed. Here, we investigated the role of a conserved hydrogen bond interaction between two helices that are suggested to undergo a large conformational change upon membrane insertion. Mutation of either the histidine or the tyrosine resulted in a protein that has significantly reduced bioactivity, increased overall flexibility, and significantly reduced stability. These data highlight an important role for this interaction in the overall stability of the protein. Additionally, the conservation of histidine and tyrosine in these positions may suggest a functional role for the interaction in the conformational switching from soluble to membrane protein. Proteins 2006.