Evidence for the prion hypothesis: induction of the yeast [PSI+] factor by in vitro- converted Sup35 protein.

Starting with purified, bacterially produced protein, we have created a [PSI(+)]-inducing agent based on an altered (prion) conformation of the yeast Sup35 protein. After converting Sup35p to its prion conformation in vitro, we introduced it into the cytoplasm of living yeast using a liposome transformation protocol. Introduction of substoichiometric quantities of converted Sup35p greatly increased the rate of appearance of the well-characterized epigenetic factor [PSI+], which results from self-propagating aggregates of cellular Sup35p. Thus, as predicted by the prion hypothesis, proteins can act as infectious agents by causing self-propagating conformational changes.

How GroES regulates binding of nonnative protein to GroEL.

At present, it is still enigmatic how the reaction cycle by which the Escherichia coli GroE chaperones mediate protein folding in the cell is coordinated with respect to the sequential order of binding and release of GroES, nucleotide, and nonnative protein. It is generally assumed that the asymmetric GroEL.GroES complex is the acceptor state for substrate protein. Nevertheless, this species is poorly understood in its binding characteristics for nucleotide and nonnative protein. We show here that this species has a high affinity binding site for nonnative protein. In addition to this, binding of nucleotide to one GroEL ring is strongly favored by GroES binding to the other ring. However, the slow rate of release of substrate protein from the unproductive trans-position kinetically favors the binding of a second GroES, thereby forming a symmetric GroEL14.(GroES7)2 complex and simultaneously ensuring that substrate protein is sequestered in a position underneath GroES. Our results demonstrate that the intrinsic binding characteristics of the trans-bullet complex determine the sequence of events during the reaction cycle.

Catalysis of protein folding by symmetric chaperone complexes.

The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way. Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved. Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions. We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way. GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold. Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL x GroES2 particles and the intermediate release of the nonnative protein from the chaperone complex. Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding.

Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants.

Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo. Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions. We used maltose-binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspecific hydrophobic interactions. Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol. Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3. However the on-rate is decreased by at least two orders of magnitude. Therefore, the rebinding reaction is prevented and folding occurs in solution.