Protein aggregation determinants from a simplified model: cooperative folders resist aggregation

Two-chain aggregation simulations using minimalist models of proteins G, L, and mutants were used to investigate the fundamentals of protein aggregation. Mutations were selected to break up repeats of hydrophobic beads in the sequence while maintaining native topology and folding ability. Data are collected under conditions in which all chain types have similar folded populations and after equilibrating the separated chains to minimize competition between folding and aggregation. Folding cooperativity stands out as the best single-chain determinant under these conditions and for these simple models. It can be experimentally measured by the width of the unfolding transition during thermal denaturation and loosely related to population of intermediate-like states during folding. Additional measures of cooperativity and other properties such as radius of gyration fluctuations and patterning of hydrophobic residues are also examined. Initial contact system states with transition-state characteristics can be identified and are more expanded than average initial contact states. Two-chain minimalist model aggregates are considerably less structured than their native states and have minimal domain-swapping features.     

Assembly of a tetrameric alpha-helical bundle: computer simulations on an intermediate-resolution protein model.

Discontinuous molecular dynamics (DMD) simulation on an intermediate-resolution protein model is used to study the folding of an isolated, small model peptide to an amphipathic alpha-helix and the assembly of four of these model peptides into a four-helix bundle. A total of 129 simulations were performed on the isolated peptide, and 50 simulations were performed on the four-peptide system. Simulations efficiently sample conformational space allowing complete folding trajectories from random initial configurations to be observed within 15 min for the one-peptide system and within 15 h for the four-peptide system on a 500-MHz workstation. The native structures of both the alpha-helix and the four-helix bundle are consistent with experimental characterization studies and with results from previous simulations on these model peptides. In both the one- and four-peptide systems, the native state is achieved during simulations within an optimal temperature range, a phenomenon also observed experimentally. The ease with which our simulations yield reasonable estimates of folded structures demonstrates the power of the intermediate-resolution model developed for this work and the DMD algorithm and suggests that simulations of very long times and of multiprotein systems may be possible with this model.     

Folding and unfolding of gammaTIM monomers and dimers

Kinetic simulations of the folding and unfolding of triosephosphate isomerase (TIM) from yeast were conducted using a single monomer gammaTIM polypeptide chain that folds as a monomer and two gammaTIM chains that fold to the native dimer structure. The basic protein model used was a minimalist Gō model using the native structure to determine attractive energies in the protein chain. For each simulation type--monomer unfolding, monomer refolding, dimer unfolding, and dimer refolding--thirty simulations were conducted, successfully capturing each reaction in full. Analysis of the simulations demonstrates four main conclusions. First, all four simulation types have a similar "folding order", i.e., they have similar structures in intermediate stages of folding between the unfolded and folded state. Second, despite this similarity, different intermediate stages are more or less populated in the four different simulations, with 1), no intermediates populated in monomer unfolding; 2), two intermediates populated with beta(2)-beta(4) and beta(1)-beta(5) regions folded in monomer refolding; 3), two intermediates populated with beta(2)-beta(3) and beta(2)-beta(4) regions folded in dimer unfolding; and 4), two intermediates populated with beta(1)-beta(5) and beta(1)-beta(5) + beta(6) + beta(7) + beta(8) regions folded in dimer refolding. Third, simulations demonstrate that dimer binding and unbinding can occur early in the folding process before complete monomer-chain folding. Fourth, excellent agreement is found between the simulations and MPAX (misincorporation proton alkyl exchange) experiments. In total, this agreement demonstrates that the computational Gō model is accurate for gammaTIM and that the energy landscape of gammaTIM appears funneled to the native state.     

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.     

Folding of bundles of alpha-helices in solution, membranes, and adsorbed overlayers

We propose a coarse-grained lattice model for Monte Carlo simulations of folding of proteins consisting of several alpha-helices. A chain representing a protein is considered to contain A and B monomers forming relatively stiff A subchains, mimicking helices, and flexible B links between these subchains, respectively. Using this model, we simulate (1) folding of four-helix proteins in solution; (2) folding of membrane proteins containing one, two, or four helices; and (3) refolding of four-helix proteins adsorbed at the liquid-solid interface. For these cases, we show typical scenarios of protein folding and refolding and study the dependence of the folding time on the chain length. Combining the latter results with those already available in the literature, we discuss the relative rates of folding of proteins belonging to different classes.     

Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta

A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.     

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.     

Molecular dynamics simulations of beta-hairpin folding

Molecular dynamics simulations of beta-hairpin folding have been carried out with a solvent-referenced potential at 274 K. The model peptide V4DPGV4 formed stable beta-hairpin conformations and the beta-hairpin ratio calculated by the DSSP algorithm was about 56% in the 50-ns simulation. Folding into beta-hairpin conformations is independent of the initial conformations. The simulations provided insights into the folding mechanism. The hydrogen bond often formed in a beta-turn first, and then propagated by forming more hydrogen bonds along the strands. Unfolding and refolding occurred repeatedly during the simulations. Both the hydrogen bonding and the hydrophobic interaction played important roles in forming the ordered structure. Without the hydrophobic effect, stable beta-hairpin conformations did not form in the simulations. With the same energy functions, the alanine-based peptide (AAQAA)3Y folded into helical conformations, in agreement with experiments. Folding into an alpha-helix or a beta-hairpin is amino acid sequence-dependent.     

Computer modeling and folding of four-helix bundles

In the context of simplified models of globular proteins, the requirements for the unique folding to a four-helix bundle have been addressed through a new Monte Carlo procedure. In particular, the relative importance of secondary versus tertiary interactions in determining the nature of the folded structure is examined. Various cases spanning the extremes where tertiary interactions completely dominate to that where tertiary interactions are negligible have been explored. Not surprisingly, the folding to unique four-helix bundles is found to depend on an adequate balance of the secondary and tertiary interactions. Moreover, because the simplified model is composed of spheres representing α-carbons and side chains, the geometry of the latter being based on small real amino acids, the role played by the side chains, and the problems associated with packing and hard-core repulsions, are considered. Also, possible folding intermediates and their relationship with the experimentally observed molten globule state are explored. From these studies, a general set of rules is extracted which should aid in the further design of more detailed protein models adequate to more fully investigate the protein folding problem. Finally, the relationship between our conclusions and experimental work with specifically designed sequences is briefly discussed. © 1993 Wiley-Liss, Inc.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β₁-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β₁-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.     

Dynamic Monte Carlo simulations of globular protein folding. Model studies of in vivo assembly of four helix bundles and four member beta-barrels

As part of an ongoing series of dynamic Monte Carlo simulations of globular protein folding, the nature of the folding pathway, of model four-member beta-barrels and four-helix bundles, under highly idealized conditions in vivo, has been examined. The ribosome is crudely modeled as an inert hard wall on to which the model protein chain is attached. Three cases are considered in detail. The first corresponds to post-translational assembly in which the fully synthesized chain is tethered to the wall and starts out under strongly denaturing conditions. The system is cooled down, and the chain is allowed to fold. Interestingly, the helical motif prefers to assemble parallel to the wall, whereas the beta-barrel, predominantly assembles with its principal axis perpendicular to the wall. In the former case, the dominant intermediate, the helical hairpin, is different from that in free solution, a three-helix bundle. The wall acts to reduce the expanse of configuration space that must be searched and aids in folding. Two situations that might lead to co-translational folding are also simulated. In the first case, to eliminate wall effects, the chain is slowly synthesized in free solution, and in the second case, it is slowly synthesized from the wall. In all cases, the chains are observed to fold post-translationally. While partially folded intermediates are observed during synthesis, they lack the stability to survive until chain synthesis is complete. The implications of these results for the folding in vivo of real protein chains is discussed, and a model of multiple domain protein folding is proposed.     

How Co-translational Folding of Multi-domain Protein Is Affected by Elongation Schedule: Molecular Simulations

Co-translational folding (CTF) facilitates correct folding in vivo, but its precise mechanism remains elusive. For the CTF of a three-domain protein SufI, it was reported that the translational attenuation is obligatory to acquire the functional state. Here, to gain structural insights on the underlying mechanisms, we performed comparative molecular simulations of SufI that mimic CTF as well as refolding schemes. A CTF scheme that relied on a codon-based prediction of translational rates exhibited folding probability markedly higher than that by the refolding scheme. When the CTF schedule is speeded up, the success rate dropped. These agree with experiments. Structural investigation clarified that misfolding of the middle domain was much more frequent in the refolding scheme than that in the codon-based CTF scheme. The middle domain is less stable and can fold via interactions with the folded N-terminal domain. Folding pathway networks showed the codon-based CTF gives narrower pathways to the native state than the refolding scheme.     

Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity, and trapping in the ribosome

Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.     

Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Protein folding in vitro and in the cellular environment

The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino-acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re-evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assisted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the "molten globule" state. However, very little is known about the recognition process.     

Dynamic Monte Carlo simulations of a new lattice model of globular protein folding, structure and dynamics

A long-standing problem of molecular biology is the prediction of globular protein tertiary structure from the primary sequence. In the context of a new, 24-nearest-neighbor lattice model of proteins that includes both alpha and beta-carbon atoms, the requirements for folding to a unique four-member beta-barrel, four-helix bundles and a model alpha/beta-bundle have been explored. A number of distinct situations are examined, but the common requirements for the formation of a unique native conformation are tertiary interactions plus the presence of relatively small (but not irrelevant) intrinsic turn preferences that select out the native conformer from a manifold of compact states. When side-chains are explicitly included, there are many conformations having the same or a slightly greater number of side-chain contacts as in the native conformation, and it is the local intrinsic turn preferences that produce the conformational selectivity on collapse. The local preference for helix or beta-sheet secondary structure may be at odds with the secondary structure ultimately found in the native conformation. The requisite intrinsic turn populations are about 0.3% for beta-proteins, 2% for mixed alpha/beta-proteins and 6% for helix bundles. In addition, an idealized model of an allosteric conformational transition has been examined. Folding occurs predominantly by a sequential on-site assembly mechanism with folding initiating either at a turn or from an isolated helix or beta-strand (where appropriate). For helical and beta-protein models, similar folding pathways were obtained in diamond lattice simulations, using an entirely different set of local Monte Carlo moves. This argues strongly that the results are universal; that is, they are independent of lattice, protein model or the particular realization of Monte Carlo dynamics. Overall, these simulations demonstrate that the folding of all known protein motifs can be achieved in the context of a single class of lattice models that includes realistic backbone structures and idealized side-chains.     

The consistency principle in protein structure and pathways of folding

• 1.i) It is pointed out that various energy terms contributing to stabilize the native state of globular proteins are consistent in the first approximation with each other in the native state. This means that each energy term is individually minimized at the minimum point of the total energy. I proposed (1) to call this fact “the consistency principle in protein structure.”
• 2.ii) The fair success of various methods of prediction of the secondary structures in globular proteins from their amino acid sequence is often interpreted as indicating the dominance of the short-range interactions in determining the local structures of the polypeptide chains. Partly from such a point of view, the hierarchic condensation model has been popular for the process of protein folding. However the consistency principle indicates that the short-range interactions are just one type of intramolecular interaction which contributes to stabilization of the native structure together with other mutually consistent types of intramolecular interactions. Therefore the hierarchic condensation model is not necessarily a unique model of protein folding.
• 3.iii) Roles of a possible nonspecific globular state, stabilized by nonspecific long-range intramolecular interactions, in the folding process are discussed. It is expected that this nonspecific globular state is observed either as an equilibrium or a kinetic intermediate state between the unfolded and the folded native states. Observation as a kinetic intermediate state is expected to occur in experiments done under strongly refolding conditions. In this case the polypeptide chain in the unfolded state collapses into a nonspecific globule by the action of nonspecific long-range intramolecular interactions. Two possible mechanisms of the transition from the nonspecific globular state to the specific native folded state are discussed.
• 4.iv) In an experiment done under weakly refolding conditions, folding is expected to occur according to the embryo-nucleus model. This model is a refined version of the hierarchic condensation model. Refinement is done by taking into account the fact that the intermediate structures assumed in the hierarchic condensation model are unstable against both the native folded state and the unfolded state. A nucleus is an ordered structure of a certain size. Ordered structures of a size larger than a nucleus tend to fold further to become the native specific globule. Ordered structures of a size smaller than a nucleus tend to unfold. Embryos are intrinsically unstable ordered structures smaller than a nucleus. Folding occurs when embryos grow in size to become a nucleus. The intrinsic instability of embryos is the built-in mechanism to overcome the low resolving power of the short-range interactions in determining local conformations of the polypeptide chain.