Mechanism of substrate recognition by the chaperonin GroEL.

The bacterial chaperonin GroEL functions with its cofactor GroES in assisting the folding of a wide range of proteins in an ATP-dependent manner. GroEL-GroES constitute one of the main chaperone systems in the Escherichia coli cytoplasm. The chaperonin facilitates protein folding by enclosing substrate proteins in a cage defined by the GroEL cylinder and the GroES cap where folding can take place in a protected environment. The in vivo role of GroEL has recently been elucidated. GroEL is found to interact with 10-15% of newly synthesized proteins, with a strong preference for proteins in the molecular weight range of 20-60 kDa. A large number of GroEL substrates have been identified and were found to preferentially contain proteins with multiple alphabeta, domains that have alpha-helices and beta-sheets with extensive hydrophobic surfaces. Based on the preferential binding of GroEL to these proteins and structural and biochemical data, a model of substrate recognition by GroEL is proposed. According to this model, binding takes place preferentially between the hydrophobic residues in the apical domains of GroEL and the hydrophobic faces exposed by the beta-sheets or alpha-helices in the alphabeta domains of protein substrates.

Chaperone-assisted protein folding in the cell cytoplasm.

Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones. Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions. There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins. Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation. Chaperones can also promote the disaggregation of preformed protein aggregates. Many of the identified chaperones are also heat shock proteins. The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides. The four main chaperone systems in the Escherichia coli cytoplasm are as follows. (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains. (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE. This system recognizes polypeptide chains in an extended conformation. (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces. (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins. These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation. Several advances have recently been made in characterizing the structure and function of all of these chaperone systems. These advances have provided us with a better understanding of the protein folding process in the cell.

Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

Identification of in vivo substrates of the chaperonin GroEL.

The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli. Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes. About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance. GroEL substrates consist preferentially of two or more domains with alphabeta-folds, which contain alpha-helices and buried beta-sheets with extensive hydrophobic surfaces. These proteins are expected to fold slowly and be prone to aggregation. The hydrophobic binding regions of GroEL may be well adapted to interact with the non-native states of alphabeta-domain proteins.

Definition of amide protection factors for early kinetic intermediates in protein folding.

Hydrogen-deuterium exchange experiments have been used previously to investigate the structures of well defined states of a given protein. These include the native state, the unfolded state, and any intermediates that can be stably populated at equilibrium. More recently, the hydrogen-deuterium exchange technique has been applied in kinetic labeling experiments to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and nonequilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent-exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. To clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one is P struc, which is more related to the structure of the intermediate, and the other is P app, which is more related to the stability of the intermediate. Kinetic hydrogen-deuterium exchange data from disulfide-intact ribonuclease A and from cytochrome c are discussed to explain the use and implications of these two definitions.

In vivo observation of polypeptide flux through the bacterial chaperonin system.

The quantitative contribution of chaperonin GroEL to protein folding in E. coli was analyzed. A diverse set of newly synthesized polypeptides, predominantly between 10-55 kDa, interacts with GroEL, accounting for 10%-15% of all cytoplasmic protein under normal growth conditions, and for 30% or more upon exposure to heat stress. Most proteins leave GroEL rapidly within 10-30 s. We distinguish three classes of substrate proteins: (I) proteins with a chaperonin-independent folding pathway; (II) proteins, more than 50% of total, with an intermediate chaperonin dependence for which normally only a small fraction transits GroEL; and (III) a set of highly chaperonin-dependent proteins, many of which dissociate slowly from GroEL and probably require sequestration of aggregation-sensitive intermediates within the GroEL cavity for successful folding.

Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.

Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.

Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization.

The equilibrium unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species. Previously, four unfolded species have been detected experimentally. They are Uvf, Uf, UsII, and UsI which have refolding time constants on the millisecond, millisecond to second, second to tens of seconds, and hundreds of seconds time scales, respectively. In the current study, the refolding pathway of the protein was investigated under favorable folding conditions of 0.58 M GdnHCl, pH 5.0, and 15 degrees C. In addition to the above four unfolded species, the presence of a fifth unfolded species was detected. It has a refolding time constant on the order of 2 s under the conditions employed. This new unfolded species is labeled Um, for medium-refolding species. Single-jump refolding experiments monitored by tyrosine burial and by cytidine 2'-monophosphate inhibitor binding indicate that the different unfolded species refold to the native state along independent refolding pathways. The buildup of the different unfolded species upon unfolding of the protein from the native state was monitored by absorbance using double-jump experiments. These experiments were carried out at 15 degrees C and consisted of an unfolding step at 4.2 M GdnHCl and pH 2.0, followed, after a variable delay time, by a refolding step at 0.58 M GdnHCl and pH 5.0. The results of these experiments support the conclusion that the different unfolded species arise from cis-trans isomerizations at the X-Pro peptide bonds of Pro 93, 114, and 117 in the unfolded state of the protein. The rates of these isomerizations were obtained for each of these three X-Pro peptide bonds at 15 degrees C.

Structure of a hydrophobically collapsed intermediate on the conformational folding pathway of ribonuclease A probed by hydrogen-deuterium exchange.

The unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species which have different X-Pro peptide bond conformations. One of these unfolded species, labeled Uvf, has all its X-Pro peptide bonds in the native conformation. Therefore, the refolding of Uvf is a purely conformational folding process which is not complicated by cis-trans X-Pro peptide bond isomerization. There are two identifiable intermediates on the folding pathway of Uvf: one which is a largely unfolded intermediate (IU) and another which is a hydrophobically collapsed intermediate (I phi). An instrument was built, and experiments were designed to study the structure in IU and I phi by hydrogen-deuterium exchange. These experiments are a combination of a double-jump experiment followed by a pulse-labeling experiment. The native protein was first unfolded to populate Uvf to more than 99%, and then Uvf was refolded for a specified period of time. After refolding, hydrogen-deuterium exchange of the backbone amides was initiated for a given time by raising the pH. Subsequently, the exchange was quenched and the protein was allowed to continue to fold to the native state. The extent of exchange was determined quantitatively by two-dimensional NMR spectroscopy. The data indicate that IU has no secondary structure that can protect the backbone amides from exchange under the conditions employed. On the other hand, in I phi, the second helix (residues 24-34) and a large part of the beta-sheet region of the protein are formed, while the rest of the protein molecule remains unstructured. In general, the protection factors in I phi are low, indicating that this intermediate has a dynamic structure. Our observations are consistent with I phi being a molten-globule-like intermediate. The regular structure formed in I phi is much less than that observed in a hydrogen-bonded intermediate (Ii) populated early on the major slow-refolding pathway of the protein [Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197-8201]; in addition, the structure in I phi has much lower stability than that in Ii. This implies that a slower refolding rate allows for a higher cooperativity between the different structural elements of the protein, resulting in the formation of more stable (native-like) intermediates (as in Ii) during the folding process.

Circular dichroism evidence for the presence of burst-phase intermediates on the conformational folding pathway of ribonuclease A.

Refolding of the very-fast-folding unfolded species (Uvf) of disulfide-intact bovine pancreatic ribonuclease A has been monitored by circular dichroism (CD) at 222 and 275 nm at 0.9 or 2.6 M guanidine hydrochloride, pH 7.0, and 5 degrees C. The refolding of Uvf represents a purely conformational folding process which is not complicated by cis-trans proline isomerization. The data indicate that there are at least two intermediates on the refolding pathway of Uvf and that both intermediates form in the burst phase when the refolding is monitored by CD. At the initiation of folding, Uvf is converted to a largely unfolded intermediate, termed Iu, which then undergoes a hydrophobic collapse to form the molten-globule-like intermediate I phi. The CD values obtained for Iu and I phi indicate that IU has no significant secondary structure and presumably differs from Uvf by a local structural rearrangement, while I phi has a substantial population of secondary and tertiary structures, about 40%-50% of that of native.

The nature of the initial step in the conformational folding of disulphide-intact ribonuclease A.

Here we investigate conformational folding reaction of disulphide-intact ribonuclease A in the absence of the complicating effects due to non-native interactions (such as cis/trans proline isomerization) in the unfolded state. The conformational folding process is found to be intrinsically very fast occurring on the milliseconds time scale. The kinetic data indicate that the conformational folding of ribonuclease A proceeds through the formation of a hydrophobically collapsed intermediate with properties similar to those of equilibrium molten-globules. Furthermore, the data suggest that the rate-limiting transition states on the unfolding and refolding pathways are substantially different with the refolding transition state having non-native-like properties.

A very fast phase in the refolding of disulfide-intact ribonuclease A: implications for the refolding and unfolding pathways.

The refolding and unfolding of disulfide-intact ribonuclease A has been studied by using single-jump and double-jump stopped-flow techniques. Absorbance and fluorescence detection methods were used to follow the kinetics. By appropriate choice of solution conditions (1.5 M guanidine hydrochloride, pH 3.0, at temperatures < or = 15 degrees C) to slow the refolding process, a new very fast phase has been observed in addition to the usual fast and slow phases that involve the unfolded species Uf and U(s), respectively. Double-jump experiments consisting of an unfolding step at 4.2 M guanidine hydrochloride and pH 2.0 followed by a refolding step at 1.5 M guanidine hydrochloride and pH 3.0 were carried out to monitor the unfolding process. These experiments demonstrated that the new phase arises from a separate unfolded species, Uvf, which is present to the extent of about 6% in the equilibrium ensemble of unfolded protein at high guanidine hydrochloride concentration and low pH. A new model for the unfolding pathway and interconversion among unfolded species is proposed based on two independent isomerization processes. The equilibrium constants and activation energies obtained for each process suggest that they involve the isomerization of cis prolines. We propose that the isomerizations occur at the X-Pro peptide bonds of Pro 93 and 114. In the model, Uvf is the first species to form without isomerization at any cis X-Pro peptide bonds when the native protein is unfolded; Uf and U(s) then form from Uvf through two independent isomerization processes. Both prolines are in the native (cis) conformation in Uvf. In Uf, Pro 114 is in a nonnative (trans) conformation while, in U(s), Pro 93 is in a nonnative (trans) conformation. The slow folding species, U(s), actually consists of (at least) two species: U(s) alpha with Pro 93 in a nonnative (trans) conformation and U(s) beta with both Pro 93 and 114 in nonnative (trans) conformations. Finally, the kinetic data suggest that the presence of a nonnative trans conformation at the Tyr 92-Pro 93 peptide bond impedes the refolding rate of ribonuclease A much more than the presence of a nonnative trans conformation at the Asn 113-Pro 114 peptide bond.