An osmolyte mitigates the destabilizing effect of protein crowding

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.     

Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another

Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.     

Folding thermodynamics of three beta-sheet peptides: a model study

We study the folding thermodynamics of a beta-hairpin and two three-stranded beta-sheet peptides using a simplified sequence-based all-atom model, in which folding is driven mainly by backbone hydrogen bonding and effective hydrophobic attraction. The native populations obtained for these three sequences are in good agreement with experimental data. We also show that the apparent native population depends on which observable is studied; the hydrophobicity energy and the number of native hydrogen bonds give different results. The magnitude of this dependence matches well with the results obtained in two different experiments on the beta-hairpin.     

Translocons, thermodynamics, and the folding of membrane proteins

Recent three-dimensional structures of helical membrane proteins present new challenges for the prediction of structure from amino acid sequence. Membrane proteins reside stably in a thermodynamic free energy minimum after release into the membrane's bilayer fabric from the translocon complex. This means that structure prediction is primarily a problem of physical chemistry. But the folding processes within the translocon must also be considered. A distilled overview of the physical principles of membrane protein stability is presented, and extended to encompass translocon-assisted folding.     

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Solvent-amino acid interaction energies in three-dimensional-lattice Monte Carlo simulations of a model 27-mer protein: Folding thermodynamics and kinetics

Amino acid residue-solvent interactions are required for lattice Monte Carlo simulations of model proteins in water. In this study, we propose an interaction-energy scale that is based on the interaction scale by Miyazawa and Jernigan. It permits systematic variation of the amino acid-solvent interactions by introducing a contrast parameter for the hydrophobicity, C(s), and a mean attraction parameter for the amino acids, omega. Changes in the interaction energies strongly affect many protein properties. We present an optimized energy parameter set for best representing realistic behavior typical for many proteins (fast folding and high cooperativity for single chains). Our optimal parameters feature a much weaker hydrophobicity contrast and mean attraction than does the original interaction scale. The proposed interaction scale is designed for calculating the behavior of proteins in bulk and at interfaces as a function of solvent characteristics, as well as protein size and sequence.     

Gatekeepers in the ribosomal protein s6: thermodynamics, kinetics, and folding pathways revealed by a minimalist protein model

We investigate the effect of structural gatekeepers on the folding of the ribosomal protein S6. Folding thermodynamics and early refolding kinetics are studied for this system utilizing computer simulations of a minimalist protein model. When gatekeepers are eliminated, the thermodynamic signature of a folding intermediate emerges, and a marked decrease in folding efficiency is observed. We explain the prerequisites that determine the "strength" of a given gatekeeper. The investigated gatekeepers are found to have distinct functions, and to guide the folding and time-dependent packing of non-overlapping secondary structure elements in the protein. Gatekeepers avoid kinetic traps during folding by favoring the formation of "productive topologies" on the way to the native state. The trends in folding rates in the presence/absence of gatekeepers observed for our minimalist model of S6 are in very good agreement with experimental data on this protein.     

The osmolyte trimethylamine‐N‐oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state

Trimethylamine‐N‐oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Φ‐value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Φ‐values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO‐induced change in the denaturant β factors. Through TMAO‐induced folding studies, a β factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.     

An all-atom structure-based potential for proteins: bridging minimal models with all-atom empirical forcefields

Protein dynamics take place on many time and length scales. Coarse-grained structure-based (Go) models utilize the funneled energy landscape theory of protein folding to provide an understanding of both long time and long length scale dynamics. All-atom empirical forcefields with explicit solvent can elucidate our understanding of short time dynamics with high energetic and structural resolution. Thus, structure-based models with atomic details included can be used to bridge our understanding between these two approaches. We report on the robustness of folding mechanisms in one such all-atom model. Results for the B domain of Protein A, the SH3 domain of C-Src Kinase, and Chymotrypsin Inhibitor 2 are reported. The interplay between side chain packing and backbone folding is explored. We also compare this model to a C(alpha) structure-based model and an all-atom empirical forcefield. Key findings include: (1) backbone collapse is accompanied by partial side chain packing in a cooperative transition and residual side chain packing occurs gradually with decreasing temperature, (2) folding mechanisms are robust to variations of the energetic parameters, (3) protein folding free-energy barriers can be manipulated through parametric modifications, (4) the global folding mechanisms in a C(alpha) model and the all-atom model agree, although differences can be attributed to energetic heterogeneity in the all-atom model, and (5) proline residues have significant effects on folding mechanisms, independent of isomerization effects. Because this structure-based model has atomic resolution, this work lays the foundation for future studies to probe the contributions of specific energetic factors on protein folding and function.     

α‐chymotrypsin in water–acetone and water–dimethyl sulfoxide mixtures: Effect of preferential solvation and hydration

We investigated water/organic solvent sorption and residual enzyme activity to simultaneously monitor preferential solvation/hydration of protein macromolecules in the entire range of water content at 25°C. We applied this approach to estimate protein destabilization/stabilization due to the preferential interactions of bovine pancreatic α‐chymotrypsin with water‐acetone (moderate‐strength H‐bond acceptor) and water‐DMSO (strong H‐bond acceptor) mixtures. There are three concentration regimes for the dried α‐chymotrypsin. α‐Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity values are close to 100%. At intermediate water content, the dehydrated α‐chymotrypsin has a higher affinity for acetone/DMSO than for water. Residual enzyme activity is minimal in this concentration range. The acetone/DMSO molecules are preferentially excluded from the protein surface at the lowest water content, resulting in preferential hydration. The residual catalytic activity in the water‐poor acetone is ～80%, compared with that observed after incubation in pure water. This effect is very small for the water‐poor DMSO. Two different schemes are operative for the hydrated enzyme. At high and intermediate water content, α‐chymotrypsin exhibits preferential hydration. However, at intermediate water content, in contrast to the dried enzyme, the initially hydrated α‐chymotrypsin possesses increased preferential hydration parameters. At low water content, no residual enzyme activity was observed. Preferential binding of DMSO/acetone to α‐chymotrypsin was detected. Our data clearly demonstrate that the hydrogen bond accepting ability of organic solvents and the protein hydration level constitute key factors in determining the stability of protein–water–organic solvent systems.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Dynamic polyhedral models of globular proteins

We have devised several mechanical models of globular proteins by approximating them to various polyhedra (dodecahedron, truncated octahedron, icosahedron, truncated icosahedron). The models comprise hollow blocks linked together in a flexible chain. Between blocks there is a set of several reversible, weak magnetic interactions such that when the chain is agitated, it will fold into a stable polyhedral structure about the size of a hand. Folding may be followed in real time with a video camera. Key to the success of the folding process is the lightness of the chain. Several side chains may also be added to the blocks such that they come together to create a polyhedral core when the chain folds. The models have a number of similarities to globular proteins: each chain folds into a unique, but dynamic, three-dimensional structure; the instructions that determine this structure are built into the configuration of blocks; and it is difficult to predict this structure given the unfolded block configuration. Furthermore, the chains fold quickly, generally in less than a minute, several pathways are involved, and these pathways progress through elements of "native" structure. In particular, the models emphasize the importance of restricted conformational mobility in assisting the chain to fold, and also in eliminating undesirable interactions. Because of these similarities to globular proteins, we believe that the polyhedral models will, with continued development, be helpful in understanding the protein folding process, while at the same time acting as valuable educational visual aids. They might also inspire the construction of new types of microscopic, self-assembling devices.     

A model study of protein nascent chain and cotranslational folding using hydrophobic-polar residues

To study protein nascent chain folding during biosynthesis, we investigate the folding behavior of models of hydrophobic and polar (HP) chains at growing length using both two-dimensional square lattice model and an optimized three-dimensional 4-state discrete off-lattice model. After enumerating all possible sequences and conformations of HP heteropolymers up to length N = 18 and N = 15 in two and three-dimensional space, respectively, we examine changes in adopted structure, stability, and tolerance to single point mutation as the nascent chain grows. In both models, we find that stable model proteins have fewer folded nascent chains during growth, and often will only fold after reaching full length. For the few occasions where partial chains of stable proteins fold, these partial conformations on average are very similar to the corresponding parts of the final conformations at full length. Conversely, we find that sequences with fewer stable nascent chains and sequences with native-like folded nascent chains are more stable. In addition, these stable sequences in general can have many more point mutations and still fold into the same conformation as the wild type sequence. Our results suggest that stable proteins are less likely to be trapped in metastable conformations during biosynthesis, and are more resistant to point-mutations. Our results also imply that less stable proteins will require the assistance of chaperone and other factors during nascent chain folding. Taken together with other reported studies, it seems that cotranslational folding may not be a general mechanism of in vivo protein folding for small proteins, and in vitro folding studies are still relevant for understanding how proteins fold biologically.     

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.     

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods (panning). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l^–1 of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.Abbreviations Ap^R ampicillin resistance - cfu colony forming unit(s) - cpIII gene III-encoded coat protein of M13 - cpVIII gene VIII-encoded coat protein of M13 - ER endoplasmic reticulum - Fab fragment of Ig containing light chain, variable region and first constant region of heavy chain - Fd variable region and first constant region of the heavy chain - Fv fragment containing variable regions of heavy and light chain - Ig immunoglobulin - Km^R kanamycin resistance - kb kilobase or 1000 basepairs - PAI-1 plasminogen activator inhibitor 1 - t-PA tissue-type plasminogen activator - u-PA urokinase-type plasminogen activator     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.