Sequence Determinants of the Aggregation of Proteins Within Condensates Generated by Liquid-liquid Phase Separation

The transition between the native and amyloid states of proteins can proceed via a deposition pathway via oligomeric intermediates or via a condensation pathway involving liquid droplet intermediates generated through liquid-liquid phase separation. While several computational methods are available to perform sequence-based predictions of the propensity of proteins to aggregate via the deposition pathway, much less is known about the physico-chemical principles that underlie aggregation within condensates. Here we investigate the sequence determinants of aggregation via the condensation pathway, and identify three relevant features: droplet-promoting propensity, aggregation-promoting propensity and multimodal interactions quantified by the binding mode entropy. By using this approach, we show that it is possible to predict aggregation-promoting mutations in droplet-forming proteins associated with amyotrophic lateral sclerosis (ALS). This analysis provides insights into the amino acid code for the conversion of proteins between liquid-like and solid-like condensates.     

Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle x-ray scattering

To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH approximately 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.     

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Thermal stability of proteins in the presence of poly(ethylene glycols)

Thermal unfolding of ribonuclease, lysozyme, chymotrypsinogen, and beta-lactoglobulin was studied in the absence or presence of poly(ethylene glycols). The unfolding curves were fitted to a two-state model by a nonlinear least-squares program to obtain values of delta H, delta S, and the melting temperature Tm. A decrease in thermal transition temperature was observed in the presence of poly(ethylene glycol) for all of the protein systems studied. The magnitude of such a decrease depends on the particular protein and the molecular size of poly(ethylene glycol) employed. A linear relation can be established between the magnitude of the decrease in transition temperature and the average hydrophobicity of these proteins; namely, the largest observable decrease is associated with the protein of the highest hydrophobicity. Further analysis of the thermal unfolding data reveals that poly(ethylene glycols) significantly effect the relation between delta H degrees of unfolding and temperature for all the proteins studied. For beta-lactoglobulin, a plot of delta H versus Tm indicates a change in slope from a negative to a positive value, thus implying a change in delta Cp in thermal unfolding caused by the presence of poly(ethylene glycols). Results from solvent-protein interaction studies indicate that at high temperature poly(ethylene glycol) 1000 preferentially interacts with the denatured state of protein but is excluded from the native state at low temperature. These observations are consistent with the fact that poly(ethylene glycols) are hydrophobic in nature and will interact favorably with the hydrophobic side chains exposed upon unfolding; thus, it leads to a lowering of thermal transition temperature.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. III: directional protein interactions

We extend our coarse-grained modeling strategy described in parts I and II of this investigation to account for nonuniform spatial distributions of hydrophobic residues on the solvent-exposed surfaces of native proteins. Within this framework, we explore how patchy surfaces can influence the solvent-mediated protein-protein interactions, and the unfolding and self-assembly behaviors of proteins in solution. In particular, we compare the equilibrium unfolding and self-assembly trends for three model proteins that share the same overall sequence hydrophobicity, but exhibit folded configurations with different solvent-exposed native-state surface morphologies. Our model provides new insights into how directional interactions can affect native-state protein stability in solution. We find that strongly-directional attractions between native molecules with patchy surfaces can help stabilize the folded conformation through the formation of self-assembled clusters. In contrast, native proteins with more uniform surfaces are destabilized by protein-protein attractions involving the denatured state. Finally, we discuss how the simulation results provide insights into the experimental solution behaviors of several proteins that display directional interactions in their native states.     

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.     

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.

Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.     

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.     

Enhanced stability of human prion proteins with two disulfide bridges

We compare the folding equilibrium of the globular domain of the human prion protein with two variants of this domain, for which an additional disulfide bond was introduced into the location where it is found in the naturally occurring doppel protein. We find that the unfolding transition midpoint of the variants is shifted toward higher denaturant concentration, indicating that the engineered disulfide bond significantly stabilizes the global protein structure. Our results further reveal that the two-disulfide variant proteins, while possessing the same global fold as the wild-type, display marked differences in their folding pathway-in particular, the absence of a characteristic alpha-helix to beta-sheet transition, which is a fundamental feature associated with misfolding of proteins into amyloid fibrils, especially in the context of prion diseases. These surprising characteristics of disulfide mutant prion proteins have important implications for the understanding of the generic aberrant processes leading to amyloid fibril formation and protein aggregation, as well as providing insight into possible therapeutic strategies.     

Aggregation suppression of proteins by arginine during thermal unfolding

Arginine has been used to suppress aggregation of proteins during refolding and purification. We have further studied in this paper the aggregation-suppressive effects of arginine on two commercially important proteins, i.e., interleukine-6 (IL-6) and a monoclonal antibody (mAb). These proteins show extensive aggregation in aqueous buffers when subjected to thermal unfolding. Arginine suppresses aggregation concentration-dependently during thermal unfolding. However, this effect was not specific to arginine, as guanidine hydrochloride (GdnHCl) at identical concentrations also was effective. While equally effective in aggregation suppression during thermal unfolding, arginine and GdnHCl differed in their effects on the structure of the native proteins. Arginine showed no apparent adverse effects on the native protein, while GdnHCl induced conformational changes at room temperature, i.e., below the melting temperature. These additives affected the melting temperature of IL-6 as well; arginine increased it concentration-dependently, while GdnHCl increased it at low concentration but decreased at higher concentration. These results clearly demonstrate that arginine suppresses aggregation via different mechanism from that conferred by GdnHCl.     

Thermodynamic characterization of an intermediate state of human growth hormone.

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0–6 M at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.     

Nonideality and protein thermal denaturation

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.     

Mechanism of Protein Hydrophobic Chromatography. Protein Unfolding and its Contribution to Effective Hydrophobicity

Abstract

Several different monomeric proteins with either one or two domains were used to study the effect of protein unfolding on effective hydrophobicity of proteins. Protein unfolding was inhibited by cross-linking with either toluene diisocyanate or glutaraldehyde. The native enzyme was much more readily bound to the hydrophobic resin than the cross-linked species. The ligand, 3-phosphoglycerate, significantly decreased the amount of phosphoglycerate kinase able to bind to octyl-Sepharose. Sucrose also caused a statistically significant decrease in protein binding to the hydrophobic resin. These studies support the concept that the degree of protein unfolding influences effective hydrophobicity as measured by retention on octyl-Sepharose.     

Mechanism for the alpha-helix to beta-hairpin transition

The aggregation of alpha-helix-rich proteins into beta-sheet-rich amyloid fibrils is associated with fatal diseases, such as Alzheimer's disease and prion disease. During an aggregation process, protein secondary structure elements-alpha-helices-undergo conformational changes to beta-sheets. The fact that proteins with different sequences and structures undergo a similar transition on aggregation suggests that the sequence nonspecific hydrogen bond interaction among protein backbones is an important factor. We perform molecular dynamics simulations of a polyalanine model, which is an alpha-helix in its native state and observe a metastable beta-hairpin intermediate. Although a beta-hairpin has larger potential energy than an alpha-helix, the entropy of a beta-hairpin is larger because of fewer constraints imposed by the hydrogen bonds. In the vicinity of the transition temperature, we observe the interconversion of the alpha-helix and beta-sheet states via a random coil state. We also study the effect of the environment by varying the relative strength of side-chain interactions for a designed peptide-an alpha-helix in its native state. For a certain range of side-chain interaction strengths, we find that the intermediate beta-hairpin state is destabilized and even disappears, suggesting an important role of the environment in the aggregation propensity of a peptide.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

Is protein unfolding the reverse of protein folding? A lattice simulation analysis.

Simulations and experiments that monitor protein unfolding under denaturing conditions are commonly employed to study the mechanism by which a protein folds to its native state in a physiological environment. Due to the differences in conditions and the complexity of the reaction, unfolding is not necessarily the reverse of folding. To assess the relevance of temperature initiated unfolding studies to the folding problem, we compare the folding and unfolding of a 125-residue protein model by Monte Carlo dynamics at two temperatures; the lower one corresponds to the range used in T -jump experiments and the higher one to the range used in unfolding simulations of all-atom models. The trajectories that lead from the native state to the denatured state at these elevated temperatures are less diverse than those observed in the folding simulations. At the lower temperature, the system unfolds through a mandatory intermediate that corresponds to a local free energy minimum. At the higher temperature, no such intermediate is observed, but a similar pathway is followed. The structures contributing to the unfolding pathways resemble most closely those that make up the "fast track" of folding. The transition state for unfolding at the lower temperature (above Tm) is determined and is found to be more structured than the transition state for folding below the melting temperature. This shift towards the native state is consistent with the Hammond postulate. The implications for unfolding simulations of higher resolution models and for unfolding experiments of proteins are discussed.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.