The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the Escherichia coli Ribosome

Background

Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.

Results

We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.

Conclusion

The ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.     

Aggregation and assembly of phage P22 temperature-sensitive coat protein mutants in vitro mimic the in vivo phenotype

Aggregation is a common side reaction in the folding of proteins which is likely due to inappropriate interactions of folding intermediates. In the in vivo folding of phage P22 coat protein, amino acid substitutions that cause a temperature-sensitive-folding (tsf) phenotype lead to the localization of the mutant coat proteins to inclusion bodies. Investigated here is the aggregation of wild-type (WT) coat protein and 3 tsf mutants of coat protein. The tsf coat proteins aggregated when refolded in vitro at high temperature. If the tsf coat proteins were refolded at 4 degrees C, they were able attain an assembly active state. WT coat protein, on the other hand, did not aggregate significantly even when folded at high temperature. The refolded tsf mutants exhibited altered secondary and tertiary structures and had an increased surface hydrophobicity, which may explain the increased propensity of their folding intermediates to aggregate.     

Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains.

The failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorhamnosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native-like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.     

Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins

Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteions.     

Relationship between the native-state hydrogen exchange and folding pathways of a four-helix bundle protein

The hydrogen exchange behavior of a four-helix bundle protein in low concentrations of denaturant reveals some partially unfolded forms that are significantly more stable than the fully unfolded state. Kinetic folding of the protein, however, is apparently two-state in the absence of the accumulation of early folding intermediates. The partially unfolded forms are either as folded as or more folded than the rate-limiting transition state and appear to represent the major intermediates in a folding and unfolding reaction. These results are consistent with the suggestion that partially unfolded intermediates may form after the rate-limiting step for small proteins with apparent two-state folding kinetics.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.     

Effect of denaturant and protein concentrations upon protein refolding and aggregation: a simple lattice model.

We present a study of the competition between protein refolding and aggregation for simple lattice model proteins. The effect of solvent conditions (i.e., the denaturant concentration and the protein concentration) on the folding and aggregation behavior of a system of simple, two-dimensional lattice protein molecules has been investigated via (dynamic Monte Carlo simulations. The population profiles and aggregation propensities of the nine most populated intermediate configurations exhibit a complex dependence on the solution conditions that can be understood by considering the competition between intra- and interchain interactions. Some of these configurations are not even seen in isolated chain simulations; they are observed to be highly aggregation prone and are stabilized primarily by the aggregation reaction in multiple-chain systems. Aggregation arises from the association of partially folded intermediates rather than from the association of denatured random-coil states. The aggregation reaction dominates over the folding reaction at high protein concentration and low denaturant concentration, resulting in low refolding yields at those conditions. However, optimum folding conditions exist at which the refolding yield is a maximum, in agreement with some experimental observations.     

Alpha-synuclein multistate folding thermodynamics: implications for protein misfolding and aggregation

Alpha-synuclein aggregation has been tightly linked with the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Despite the protein's putative function in presynaptic vesicle regulation, the roles of lipid binding in modulating alpha-synuclein conformations and the aggregation process remain to be fully understood. This study focuses on a detailed thermodynamic characterization of monomeric alpha-synuclein folding in the presence of SDS, a well-studied lipid mimetic. Far-UV CD spectroscopy was employed for detection of conformational transitions induced by SDS, temperature, and pH. The data we present here clearly demonstrate the multistate nature of alpha-synuclein folding, which involves two predominantly alpha-helical partially folded thermodynamic intermediates that we designate as F (most folded) and I (intermediately folded) states. Likely structures of these alpha-synuclein conformational states are also discussed. These partially folded forms can exist in the presence of either monomeric or micellar forms of SDS, which suggests that alpha-synuclein has an intrinsic propensity for adopting multiple alpha-helical structures even in the absence of micelle or membrane binding, a feature that may have implications for its biological activity and toxicity. Additionally, we discuss the relation between alpha-synuclein three-state folding and its aggregation, within the context of isothermal titration calorimetry and transmission electron microscopy measurements of SDS-initiated oligomer formation.     

Reversible folding of cysteine-rich metallothionein by an overcritical reaction path

A first-order-like state transition is considered to be involved in the restoration of the activities of a few proteins by correctly folding the protein [Phys. Rev. E 66 (2002) 021903]. In order to understand the general applicability of this mechanism, we studied a metallothionein (MT) protein with an unconventional structure, i.e., without any alpha-helix or beta-sheet. MT is a 61 amino-acid peptide. There are 6-7 Zn(2+) ions, which bind avidly to 20 conserved cysteines (Cys) of MT. These properties indicate that the structure of MT is quite different from those of the other proteins. Similar to our previous findings, the denatured MT can be folded without any aggregation via a designated stepwise quasi-static process (an over-critical reaction path). The particle size of folded MT intermediates, determined by dynamic light scattering, shrank right after the first folding stage. It is consistent with a collapse-model. In addition, results from both atomic absorption and circular dichroism (CD) indicate that the stable intermediates may fold to the native conformation but with only partial Zn(2+) binding, which in turn implies that those folding intermediates are in a molten globular state. These reversible unfolding and folding processes indicate that Cys-rich protein, MT, may also be folded by way of a first-order-like state transition mechanism. We suspect that this process may likely be involved in the reaction of the metal substitution process in metal containing enzymes.     

Protein folding and aggregation: two sides of the same coin in the condensation of proteins revealed by pressure studies

Hydrostatic pressure can be considered as "thermodynamic tweezers" to approach the protein folding problem and to study the cases when folding goes wrong leading to the protein folding disorders. The main outcome of the use of high pressure in this field is the stabilization of folding intermediates such as partially folded conformations, thus allowing us to characterize their structural properties. Because partially folded intermediates are usually at the intersection between productive and off-pathway folding, they may give rise to misfolded proteins, aggregates and amyloids that are involved in many neurodegenerative diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease and Huntington's disease. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform, the PrP(Sc) (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. The last findings on the application of high pressure on amyloidogenic proteins will be discussed here as regards to their energetic and volumetric properties. The use of high pressure promises to contribute to the identification of the underlying mechanisms of these neurodegenerative diseases and to develop new therapeutic approaches.     

Cracking the folding code. Why do some proteins adopt partially folded conformations, whereas other don't?

Many, but not all, globular proteins have been shown to have compact intermediate state(s) under equilibrium conditions in vitro, giving rise to the question: why do some proteins adopt partially folded conformations, whereas other do not? Here we show that charge to hydrophobicity ratio of a polypeptide chain may represent a key determinant in this respect, as proteins known to form equilibrium partially folded intermediates are specifically localized within a unique region of charge-hydrophobicity space. Thus, the competence of a protein to form equilibrium intermediate(s) may be determined by the bulk content of hydrophobic and charged amino acid residues rather than by the positioning of amino acids within the sequence.     

Substrate-induced activation of a trapped IMC-mediated protein folding intermediate

While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.     

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

Glycine betaine: A widely reported osmolyte induces differential and selective conformational stability and enhances aggregation in some proteins in the presence of surfactants

In this study, we extensively report the effect of glycine betaine during the refolding of partially folded bovine α‐lactalbumin (α‐LA) in presence of hexadecyl trimethyl ammonium bromide (HTAB), and Ribonuclease A (RNAse A) in presence of sodium dodecyl sulfate (SDS) by different complementary biophysical, light scattering, and microscopic techniques. Though a substantial refolding/compaction was observed in both the studied proteins, the fluorescence studies contradicted the finding obtained from circular dichroism spectroscopy (CD) in case of α‐LA. CD stopped flow showed extensive presence of intermediates during the refolding of proteins which could potentially lead to aggregation. The aggregates as observed in dynamic light scattering (DLS), in α‐LA were massive as compared to RNAse A and was directly proportional to betaine concentration. The zeta potential confirmed that the aggregates are a direct manifestation of strong aggregating and/or immense preferential excluding tendency of GB and not because of charge neutralization; however a possible role of conformational change as observed in FTIR spectroscopy cannot be completely ruled out. In contrary though RNAse A showed a substantial refolding, the final state of the folded protein was significantly different from the native state. These findings for α‐LA and RNAse A were further supported by electron microscopic and thermodynamic studies. We thus propose that betaine has a strong macromolecular excluding tendency, primarily directed to shield the hydrophobic exposure either by refolding or aggregation, and depending on the hydrophobicity of the proteins, the functional restoration of the protein is manifested. © 2012 Wiley Periodicals, Inc. Biopolymers 97:933–949, 2012.     

Studying the folding process of the acylphosphatase from Sulfolobus solfataricus. A comparative analysis with other proteins from the same superfamily

The folding process of the acylphosphatase from Sulfolobus solfataricus (Sso AcP) has been followed, starting from the fully unfolded state, using a variety of spectroscopic probes, including intrinsic fluorescence, circular dichroism, and ANS binding. The results indicate that an ensemble of partially folded or misfolded species form rapidly on the submillisecond time scale after initiation of folding. This conformational ensemble produces a pronounced downward curvature in the Chevron plot, appears to possess a content of secondary structure similar to that of the native state, as revealed by far-UV circular dichroism, and appears to have surface-exposed hydrophobic clusters, as indicated by the ability of this ensemble to bind to 8-anilino-1-naphthalenesulfonic acid (ANS). Sso AcP folds from this conformational state with a rate constant of ca. 5 s(-1) at pH 5.5 and 37 degrees C. A minor slow exponential phase detected during folding (rate constant of 0.2 s(-1) under these conditions) is accelerated by cyclophilin A and is absent in a mutant of Sso AcP in which alanine replaces the proline residue at position 50. This indicates that for a lower fraction of Sso AcP molecules the folding process is rate-limited by the cis-trans isomerism of the peptide bond preceding Pro50. A comparative analysis with four other homologous proteins from the acylphosphatase superfamily shows that sequence hydrophobicity is an important determinant of the conformational stability of partially folded states that may accumulate during folding of a protein. A low net charge and a high propensity to form alpha-helical structure also emerge as possibly important determinants of the stability of partially folded states. A significant correlation is also observed between folding rate and hydrophobic content of the sequence within this superfamily, lending support to the idea that sequence hydrophobicity, in addition to relative contact order and conformational stability of the native state, is a key determinant of folding rate.     

Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) provides a unique folding environment that is distinct from other organelles supporting protein folding. The relatively oxidizing milieu allows the formation of disulfide bonds. N-linked oligosaccharides that are attached during synthesis play multiple roles in the folding process of glycoproteins. They stabilize folded domains and increase protein solubility, which prevents aggregation of folding intermediates. Glycans mediate the interaction of newly synthesized glycoproteins with some resident ER folding factors, such as calnexin and calreticulin. Here we present an overview of the present knowledge on the folding process of the heavily glycosylated human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein in the ER.