Stability Of Native State Research Articles

Protein Folding Mediated by an Intramolecular Chaperone: Energy Landscape for Unimolecular Pro-Subtilisin E Maturation

Efficient and precise assembly of polypeptides into native functional states is critical for normal cellular processes. Understanding how a specific structure is encoded in the polypeptide sequence and what drives the structural progression to the native state is essential to deciphering the folding problem. Several prokaryotic and eukaryotic proteins require their propeptide-domains to function as dedicated intramolecular chaperones (IMCs). In this manuscript, we investigate the elementary steps in the IMC mediated maturation of Subtilisin E, a bacterial serine protease, and a prototype for the eukaryotic proprotein convertases (PCs). Through detailed analyses, we have attempted to define the unimolecular folding energy landscape for SbtE to understand how the stabilization of folding intermediates influences the maturation process, an aspect that is difficult to study in eukaryotic PCs. Our studies demonstrate that a rapid hydrophobic collapse precedes acquisition of tertiary structure during the folding of Pro-SbtE and results in formation of a molten-globule like intermediate. Induction of structure within the IMC stabilizes both the molten globule-like folding intermediate and the native state, and appears to expedite initial stages of folding, purely through thermodynamic stabilization of the folded state. While the induced structure does not affect the activation energies in the unimolecular folding reaction, it is detrimental to the autoproteolytic cleavage of the precursor and subsequent release and degradation of the inhibitory IMC-domain since both these stages require some degree of unfolding. Completion of Pro-SbtE maturation results in the formation of a kinetically trapped and extremely stable native state. Hence, our results suggest that the SbtE IMC appears to have evolved to be intrinsically unstructured and to bind with its cognate protease with a specific affinity that is critical for biological regulation.

Macromolecular crowding decelerates aggregation of a β-rich protein, bovine carbonic anhydrase: a case study.

The majority of in vitro investigations concerning protein aggregation have been performed in dilute systems, which poorly reflect the crowded in vivo scenario. Cell interior is highly crowded with soluble and insoluble macromolecules that alter macromolecular properties. Macromolecular crowding is known to enhance the rate and/or extent of protein aggregation. However, most of the understandings were derived from studies with α-rich or predominantly α-proteins. Indeed, α-proteins fold faster than β-proteins and conversion of α-helices to cross β-sheets are responsible for aggregate/amyloid formation. Therefore, it is important to investigate how macromolecular crowding affects the aggregation propensity of β-rich proteins. In this study, we investigated the effect of synthetic macromolecular crowders on bovine carbonic anhydrase (BCA, a β-rich protein) aggregation. In contrast to the effect of macromolecular crowding on α-rich proteins, BCA aggregation was observed to be reduced due to decrease in the population of aggregation-prone intermediates as a consequence of increased native state stability. In addition, the extent of aggregation was found to depend on the nature of the crowder under consideration. Combining the published data on α-proteins and this study, we conclude that macromolecular crowding can have opposite consequences on protein aggregation process depending on the fold type of the protein.

Residue-specific force field based on the protein coil library. RSFF1: modification of OPLS-AA/L.

Traditional protein force fields use one set of parameters for most of the 20 amino acids (AAs), allowing transferability of the parameters. However, a significant shortcoming is the difficulty to fit the Ramachandran plots of all AA residues simultaneously, affecting the accuracy of the force field. In this Feature Article, we report a new strategy for protein force field parametrization. Backbone and side-chain conformational distributions of all 20 AA residues obtained from protein coil library were used as the target data. The dihedral angle (torsion) potentials and some local nonbonded (1-4/1-5/1-6) interactions in OPLS-AA/L force field were modified such that the target data can be excellently reproduced by molecular dynamics simulations of dipeptides (blocked AAs) in explicit water, resulting in a new force field with AA-specific parameters, RSFF1. An efficient free energy decomposition approach was developed to separate the corrections on ϕ and ψ from the two-dimensional Ramachandran plots. RSFF1 is shown to reproduce the experimental NMR (3)J-coupling constants of AA dipeptides better than other force fields. It has a good balance between α-helical and β-sheet secondary structures. It can successfully fold a set of α-helix proteins (Trp-cage and Homeodomain) and β-hairpins (Trpzip-2, GB1 hairpin), which cannot be consistently stabilized by other state-of-the-art force fields. Interestingly, the RSFF1 force field systematically overestimates the melting temperature (and the stability of native state) of these peptides/proteins. It has a potential application in the simulation of protein folding and protein structure refinement.

The βγ-crystallins: Native state stability and pathways to aggregation

The βγ-crystallins are among the most stable and long-lived proteins in the human body. With increasing age, however, they transform to high molecular weight light-scattering aggregates, resulting in cataracts. This occurs despite the presence in the lens of high concentrations of the a-crystallin chaperones. Aggregation of crystallins can be induced in vitro by a variety of stresses, including acidic pH, ultraviolet light, oxidative damage, heating or freezing, and specific amino acid substitutions. Accumulating evidence points to the existence of specific biochemical pathways of protein: protein interaction and polymerization. We review the methods used for studying crystallin stability and aggregation and discuss the sometimes counterintuitive relationships between factors that favor native state stability and those that favor non-native aggregation. We discuss the behavior of βγ-crystallins in mixtures and their chaperone ability; the consequences of missense mutations and covalent damage to the side-chains; and the evolutionary strategies that have shaped these proteins. Efforts are ongoing to reveal the nature of cataractous crystallin aggregates and understand the mechanisms of aggregation in the context of key models of protein polymerization: amyloid, native-state, and domain-swapped. Such mechanistic understanding is likely to be of value for the development of therapeutic interventions and draw attention to unanswered questions about the relationship between a protein’s native state stability and its transformation to an aggregated state.

Altered native stability is the dominant basis for susceptibility of α1-antitrypsin mutants to polymerization.

Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process.

Why Do Tetrapropylammonium Chloride and Sulphate Salts Destabilize the Native State of Globular Proteins?

It has recently been shown that aqueous solutions of tetrapropylammonium chloride and sulphate salts destabilize the folded conformation of Trp-peptides (Dempsey et al., 2011). This result is rationalized by the application of a statistical thermodynamic approach (Graziano, 2010). It is shown that the magnitude of the solvent-excluded volume effect, the main contribution for the native state stability, decreases in both aqueous 2 M TPACl solution and aqueous 1 M TPA2SO4 solution. This happens because TPA+ ions are so large in size and interact so weakly with water molecules, due to their very low charge density, to be able to counteract the electrostrictive effect of chloride and sulphate ions on the water structure, so that the density of their aqueous solutions is smaller or only slightly larger than that of water.

Posttranslational Modifications as Modulators of Mechanical Protein Folding

Understanding the molecular basis of muscle elasticity is an important challenge in medicine. For example, mutations in the giant elastic protein titin are implicated in the etiology of dilated cardiomyopathies. We use single molecule force-clamp spectroscopy to study the folding and unfolding dynamics of single polyproteins composed of tandem repeats of titin Ig domains. Protein elasticity is due to the diffusional extension and collapse of a polyprotein along its free energy landscape. Using single molecule force-clamp traces we calculate the free energy of a polyprotein along its stretching coordinate under different mechanical force regimes. At high stretching forces the free energy is dominated by changes in entropy as the molecule unfolds and extends. When the force is quenched, enthalpic interactions become important as the polyprotein slowly progresses from collapsed states to molten globules and then to the mechanically stable native state. We also show that while the polyprotein is in the extended state, cysteine residues that are normally buried become exposed to the solution where they can be post-translationally modified. We have found that S-glutathionylation of such cryptic cysteines greatly decreases the mechanical stability of an Ig domain as well as its ability to fold, favoring more extensible states in titin. Thus, the free energy landscape of a protein extending and folding under force is strongly modulated by the redox state of a cell. We propose that posttranslational modification of cryptic cysteine residues is a major regulatory pathway of tissue elasticity. Accurate predictions of elastic phenotypes in titin will be possible after we fully understand how cellular chemistry modulates the physics of a polypeptide extending under force.

Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.

Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients.

Mapping the structure of amyloid nucleation precursors by protein engineering kinetic analysis

Understanding the early molecular mechanisms governing amyloid aggregation is crucial to learn how to prevent it. Here, we used a site-directed mutagenesis approach to explore the molecular mechanism of nucleation of amyloid structure in the N47A Spc-SH3 domain. The changes in the native state stability produced by a series of mutations on each structural element of the domain were uncorrelated with the changes in the aggregation rates, although the overall aggregation mechanism was not altered. Analysis of the thioflavin T initial rates based on a simple kinetic model allowed us to extract thermodynamic magnitudes of the precursor states of nucleation and map the regions of the protein participating in the structure of the amyloidogenic precursors. This structure differs from that of the folding transition state of the SH3 domains, strongly suggesting that the regions of the conformational landscape leading to amyloid formation are divergent from those leading to the native fold.

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.

Funneling and frustration in the energy landscapes of some designed and simplified proteins.

We explore the similarities and differences between the energy landscapes of proteins that have been selected by nature and those of some proteins designed by humans. Natural proteins have evolved to function as well as fold, and this is a source of energetic frustration. The sequence of Top7, on the other hand, was designed with architecture alone in mind using only native state stability as the optimization criterion. Its topology had not previously been observed in nature. Experimental studies show that the folding kinetics of Top7 is more complex than the kinetics of folding of otherwise comparable naturally occurring proteins. In this paper, we use structure prediction tools, frustration analysis, and free energy profiles to illustrate the folding landscapes of Top7 and two other proteins designed by Takada. We use both perfectly funneled (structure-based) and predictive (transferable) models to gain insight into the role of topological versus energetic frustration in these systems and show how they differ from those found for natural proteins. We also study how robust the folding of these designs would be to the simplification of the sequences using fewer amino acid types. Simplification using a five amino acid type code results in comparable quality of structure prediction to the full sequence in some cases, while the two-letter simplification scheme dramatically reduces the quality of structure prediction.

Structural evidence for native state stabilization of a conformationally labile amyloidogenic transthyretin variant by fibrillogenesis inhibitors

Several classes of chemicals are able to bind to the thyroxine binding sites of transthyretin (TTR), stabilizing its native state and inhibiting in vitro the amyloidogenic process. The amyloidogenic I84S TTR variant undergoes a large conformational change at moderately acidic pH. Structural evidence has been obtained by X-ray analysis for the native state stabilization of I84S TTR by two chemically distinct fibrillogenesis inhibitors. In fact, they fully prevent the acidic pH-induced protein conformational change as a result of a long-range stabilizing effect. This study provides further support to the therapeutic strategy based on the use of TTR stabilizers as anti-amyloidogenic drugs. Structured summary of protein interactionsTTR and TTRbind by x-ray crystallography (View interaction)

Reduced Native State Stability in Crowded Cellular Environment Due to Protein–Protein Interactions

The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

α1-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α1-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α1-Antitrypsin has never been examined. We have therefore created a range of α1Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α1-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

The functional and structural adaptations to temperature have been addressed in homologous chloride-dependent α-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated α-amylases.

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Optimizing Solute–Water van der Waals Interactions To Reproduce Solvation Free Energies

An accurate representation of solute-water interactions is necessary for molecular dynamics simulations of biomolecules that reside in aqueous environments. Modern force fields and advanced water models describe solute-solute and water-water interactions reasonably accurately but have known shortcomings in describing solute-water interactions, demonstrated by the large differences between calculated and experimental solvation free energies across a range of peptide and drug chemistries. In this work, we introduce a method for optimizing solute-water van der Waals interactions to reproduce experimental solvation free energy data and apply it to the optimization of a fixed charge force field (AMBER ff99SB/GAFF) and advanced water model (TIP4P-Ew). We show that, with these optimizations, the combination of AMBER ff99SB/GAFF and TIP4P-Ew is able to reproduce the solvation free energies of a variety of biologically relevant small molecules to within 1.0 k(B)T. We further validate these optimizations by examining the aggregation propensities of dipeptide-water solutions, the conformational preferences of short disordered peptides, and the native state stability and dynamics of a folded protein.

E-Cadherin Destabilization Accounts for the Pathogenicity of Missense Mutations in Hereditary Diffuse Gastric Cancer

E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo.

Context-Dependent Effects of Asparagine Glycosylation on Pin WW Folding Kinetics and Thermodynamics

In extracting folding and unfolding rate information from our apparent rate constant data, and in fitting the extracted folding rates as a function of temperature to Kramer's model, we inadvertently used the pre-jump equilibrium temperature instead of the post-jump temperature: a difference of 12 °C. The absolute folding and unfolding rate values for each WW variant in Tables 3 and ​and44 are different than reported in the original paper by 2-fold, but most of the folding and unfolding rate ratios (comparing the folding or unfolding rates of the glycosylated vs. non-glycosylated WW variants) are similar to the values presented in the original paper. The changes to the data do not change the conclusion of our paper that specific, evolved protein-glycan contacts must also play a role in mediating the beneficial energetic effects on protein folding that glycosylation can confer. Table 3 Experimentally Measured and Computationally Predicted Folding Rates for Pin WW Variants Having Either Asn or Asn-GlcNAc at the Indicated Positionsa Table 4 Experimentally Measured and Computationally Predicted Unfolding Rates for Pin WW Variants Having Either Asn or Asn-GlcNAc at the Indicated Positionsa The corrected data appear in Tables 3 and ​and44 below. The corrected versions of Figures S44 to S55 (the data that Tables 3 and ​and44 summarize) are provided in the Supporting Information. The following sentences in the Results section text referring to our kinetic data (pp. 15364): The modest stabilizing effect of the Asn to Asn-GlcNAc substitution at positions 20 and 30 appears to be a result, in part, of an increased folding rate. Glycosylation at position 20 (compare 20 with 20g) increases the folding rate ⍰1.1-fold (Table 3). Glycosylation at position 30 (compare 30 with 30g) has a similar effect. These small folding rate increases agree with the predictions of the computational model and could be consistent with a small amount of denatured-state destabilization as a consequence of glycosylation. However, the decreased unfolding rate of 20g relative to 20 (also predicted by the model) could indicate that glycosylation at position 20 actually stabilizes the folding transition state and native state simultaneously. are changed to: The modest stabilizing effect of the Asn to Asn-GlcNAc substitution at position 20 appears to be primarily due to an increased folding rate (20g folds 1.5 times faster than 20). This folding rate increase agrees with the predictions of the computational model, and could be consistent with a small amount of denatured-state destabilization as a consequence of glycosylation. However, the unfolding rates of 20g and 20 are indistinguishable, which disagrees with the predicted decrease in unfolding rate upon glycosylation. The stabilizing effect of glycosylation at position 30 appears to come primarily from a decrease in unfolding rate (30g unfolds 0.7 times as fast as non-glycosylated Pin WW), as predicted by the model. This decrease compensates for an unexpected decrease in folding rate: 30g folds 0.8 times as fast as Pin WW whereas the model predicted a large increase in folding rate. Results for 33 and 33g are similarly inconsistent with the predictions of the model. In addition, the following sentence in the Discussion section text about our kinetic data (pp. 15366): For example, the observed increased stability and increased folding rate of 20g relative to 20, and of 30g relative to 30, could be the result of simultaneous transition state and native state stabilization (rather than destabilization of the denatured ensemble), reflecting the presence of favorable glycan-protein contacts at these positions. is changed to: For example, the observed increased stability of 20g and 30g relative to 20 and 30, respectively, could be the result of favorable native-state GlcNAc-protein contacts at these positions.

Effects of pH on Proteins: Predictions for Ensemble and Single-Molecule Pulling Experiments

Protein conformations change among distinct thermodynamic states as solution conditions (temperature, denaturants, pH) are altered or when they are subjected to mechanical forces. A quantitative description of the changes in the relative stabilities of the various thermodynamic states is needed to interpret and predict experimental outcomes. We provide a framework based on the Molecular Transfer Model (MTM) to account for pH effects on the properties of globular proteins. The MTM utilizes the partition function of a protein calculated from molecular simulations at one set of solution conditions to predict protein properties at another set of solution conditions. To take pH effects into account, we utilized experimentally measured pK(a) values in the native and unfolded states to calculate the free energy of transferring a protein from a reference pH to the pH of interest. We validate our approach by demonstrating that the native-state stability as a function of pH is accurately predicted for chymotrypsin inhibitor 2 (CI2) and protein G. We use the MTM to predict the response of CI2 and protein G subjected to a constant force (f) and varying pH. The phase diagrams of CI2 and protein G as a function of f and pH are dramatically different and reflect the underlying pH-dependent stability changes in the absence of force. The calculated equilibrium free energy profiles as functions of the end-to-end distance of the two proteins show that, at various pH values, CI2 unfolds via an intermediate when subjected to f. The locations of the two transition states move toward the more unstable state as f is changed, which is in accord with the Hammond-Leffler postulate. In sharp contrast, force-induced unfolding of protein G occurs in a single step. Remarkably, the location of the transition state with respect to the folded state is independent of f, which suggests that protein G is mechanically brittle. The MTM provides a natural framework for predicting the outcomes of ensemble and single-molecule experiments for a wide range of solution conditions.