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.     

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.     

The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms

Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topology but altered regional stability, using a protein engineering approach. Our data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. Our results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.     

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.     

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.     

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.     

Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.

The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.     

Disorder-to-order conformational transitions in protein structure and its relationship to disease

Function in proteins largely depends on the acquisition of specific structures through folding at physiological time scales. Under both equilibrium and non-equilibrium states, proteins develop partially structured molecules that being intermediates in the process, usually resemble the structure of the fully folded protein. These intermediates, known as molten globules, present the faculty of adopting a large variety of conformations mainly supported by changes in their side chains. Taking into account that the mechanism to obtain a fully packed structure is considered more difficult energetically than forming partially “disordered” folding intermediates, evolution might have conferred upon an important number of proteins the capability to first partially fold and—depending on the presence of specific partner ligands—switch on disorder-to-order transitions to adopt a highly ordered well-folded state and reach the lowest energy conformation possible. Disorder in this context can represent segments of proteins or complete proteins that might exist in the native state. Moreover, because this type of disorder-to-order transition in proteins has been found to be reversible, it has been frequently associated with important signaling events in the cell. Due to the central role of this phenomenon in cell biology, protein misfolding and aberrant disorder-to-order transitions have been at present associated with an important number of diseases.     

The effect of disease-associated mutations on the folding pathway of human prion protein

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.     

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.     

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.     

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.     

Photo-CIDNP NMR methods for studying protein folding

Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly powerful in time-resolved NMR measurements. Real-time, rapid-injection protein refolding experiments permit the observation of changes in the accessibility of specific residues during the folding process. CIDNP pulse-labeling gives information on the accessibility of residues in partially structured proteins (e.g., molten globule states) whose NMR spectra are broad and poorly resolved. Heteronuclear two-dimensional (15)N-(1)H CIDNP techniques allow identification of surface-accessible residues with improved resolution and sensitivity. These methods offer residue-specific structural and kinetic information on transient folding intermediates and other partially folded states of proteins that are not readily available from more routine NMR techniques.     

Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein.     

Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding

Applications of sensitive new technologies, in particular, two-dimensional NMR spectroscopy, have allowed detection of folded structures in short peptide fragments of proteins in aqueous solution under conditions where native proteins fold. These structures are in rapid dynamic exchange with unfolded states. These observations provide evidence in support of models for protein folding which postulate localized regions of folded structure as initiation sites for the folding process. Since these initiation processes are expected to be rapid, such models are consistent with kinetic evidence that the rate-determining steps of protein folding occur late in the process and probably involve rearrangement of incorrectly folded intermediates.     

Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies

The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.     

The folding process of apomyoglobin

Apomyoglobin (apoMb) folds through at least two partially folded forms that are detected both as transient intermediates during folding/unfolding kinetics or as stable intermediates at equilibrium. Here, I summarize the results of recent kinetic studies, which combined with detailed characterizations of equilibrium forms of the protein, provide a very detailed picture of apoMb folding process. The data are consistent with a linear U<->Ia<->Ib<->N model where compaction and structure are progressively acquired.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

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.