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Coiled coils are among the most abundant tertiary and quaternary structures found in proteins. A growing body of evidence suggests that long-range synergistic interactions among solvent-exposed residues can contribute substantially to coiled-coil conformational stability, but our understanding of the key sequence and structural prerequisites of this effect is still developing. Here we show that the strength of synergistic interaction involving a b-position Glu (i), an f-position Tyr (i+4), and a c-position Lys (i+8) depends on the identity of the f-position residue, the length and stability of the coiled coil, and its oligomerization stoichiometry/surface accessibility. Combined with previous observations, these results map out predictable sequence- and structure-based criteria for enhancing coiled-coil stability by up to −0.58 kcal/mol per monomer (or −2.32 kcal/mol per coiled-coil tetramer). Our observations expand the available tools for enhancing coiled coil stability by sequence variation at solvent-exposed b-, c-, and f-positions and suggest the need to exercise care in the choice of substitutions at these positions for application specific purposes.

In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a promising approach to kinetically trap, resolve, and quantify protein conformers that evolve during unfolding in solution. We conducted acid-induced unfolding of three model proteins (cytochrome

The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and

The Atlantic salmon Salmo salar survives below 10 °C. The main skeletal muscle is composed of a single isoform of tropomyosin (classified as Tpm1 α-fast) that is >92% identical to the mammalian homologue. How salmon Tpm1 maintains flexibility is investigated by reversing the only full charge substitution; threonine-77(g) in salmon and lysine in other vertebrates. The mutation (Thr-77 to Lys), which falls within a known destabilizing alanine cluster, (i) yields a useful electrophoretic shift in the absence and presence of an anionic detergent, (ii) increases the Tms of both cooperative transitions (calorimetry, 0.1 M salt, pH 7) [35 °C (minor) and 44 °C (major); ΔTm1 = 5 °C, ΔTm2 = 3.5 °C], (iii) increases the Tm of CN1A (residues 11-127) to 53 °C (ΔTm = 13 °C), a value similar to that of mammalian CN1A, (iv) markedly reduces the rate of proteolysis at Leu-169, and (v) weakens the affinity of salmon Tpm1 for troponin-Sepharose. Glu-82(e), the interstrand ionic partner of Lys-77(g), is conserved. The change in ionic interactions at this locus is postulated to be "sensed" in actin period 5 (residues 166-207) and likely beyond. Wild type (acetylated) salmon Tmp1 binds more tightly to F-actin at 4 °C than at 22 °C, which is the opposite of the long-known relationship displayed by the mammalian homologue. All of the evidence indicates that the presence of a neutral 77th amino acid destabilizes a sizable portion of salmon Tpm1 that includes the midregion. Threonine-77 is a key factor in rescuing the thin filament from the peril of cold-induced rigidity.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H–15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.

Rhodopseudomonas palustris cytochrome c', a four-helix bundle, and the second ubiquitin-associated domain, UBA(2), a three-helix bundle from the human homologue of yeast Rad23, HHR23A, deviate from random coil behavior under denaturing conditions in a fold-specific manner. The random coil deviations in each of these folds occur near interhelical turns and loops in their tertiary structures. Here, we examine an additional three-helix bundle with an identical fold to UBA(2), but a highly divergent sequence, the first ubiquitin-associated domain, UBA(1), of HHR23A. We use histidine-heme loop formation methods, employing eight single histidine variants, to probe for denatured state conformational bias of a UBA(1) domain fused to the N-terminus of iso-1-cytochrome c (iso-1-Cytc). Guanidine hydrochloride (GuHCl) denaturation shows that the iso-1-Cytc domain unfolds first, followed by the UBA(1) domain. Denatured state (4 and 6 M GuHCl) histidine-heme loop formation studies show that as the size of the histidine-heme loop increases, loop stability decreases, as expected for the Jacobson-Stockmayer relationship. However, loops formed with His35, His31, and His15, of UBA(1), are 0.6-1.1 kcal/mol more stable than expected from the Jacobson-Stockmayer relationship, confirming the importance of deviations of the denatured state from random coil behavior near interhelical turns of helical domains for facilitating folding to the correct topology. For UBA(1) and UBA(2), hydrophobic clusters on either side of the turns partially explain deviations from random coil behavior; however, helix capping also appears to be important.

The mechanisms leading to aggregation of the crystallin proteins of the eye lens remain largely unknown. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics of γD-crystallin and its cataract-related W42R variant at both infinite dilution and physiologically relevant concentrations. We find that the W42R variant assumes a distinct conformation in solution that leaves the Greek key domains of the native fold largely unaltered but lacks the hydrophobic interdomain interface that is key to the stability of wild-type γD-crystallin. At physiologically relevant concentrations, exposed hydrophobic regions in this alternative conformation become primary sites for enhanced interprotein interactions leading to large-scale aggregation.

Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, U(T) and U(D) for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone−backbone interactions and identity versus backbone−solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients.

Evidence of physical interaction with the target protein is essential in the development of chemical probes and drugs. The cellular thermal shift assay (CETSA) allows evaluation of drug binding in live cells but lacks a framework to support quantitative interpretations and comparisons with functional data. We outline an experimental platform for such analysis using human kinase p38α. Systematic variations to the assay's characteristic heat challenge demonstrate an apparent loss of compound potency with an increase in duration or temperature, in line with expectations from the literature for thermal shift assays. Importantly, data for five structurally diverse inhibitors can be quantitatively explained using a simple model of linked equilibria and published binding parameters. The platform further distinguishes between ligand mechanisms and allows for quantitative comparisons of drug binding affinities and kinetics in live cells and lysates. We believe this work has broad implications in the appropriate use of the CETSA for target and compound validation.

Cold unfolding of proteins is predicted by the Gibbs-Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium- and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (

Previous work with the four-helix-bundle protein cytochrome c' from Rhodopseudomonas palustris using histidine-heme loop formation methods revealed fold-specific deviations from random coil behavior in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix-bundle polypeptide, the second ubiquitin-associated domain, UBA(2), of the human DNA excision repair protein. We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain at the N-terminus of iso-1-cytochrome c. We have engineered histidine into highly solvent accessible positions of UBA(2), creating six single histidine variants. Guanidine hydrochloride denaturation studies show that the UBA(2)-cytochrome c fusion protein unfolds in a three-state process with iso-1-cytochrome c unfolding first. Furthermore, engineered histidine residues in UBA(2) strongly destabilize the iso-1-cytochrome c domain. Equilibrium and kinetic histidine-heme loop formation measurements in the denatured state at 4 and 6 M guanidine hydrochloride show that loop stability decreases as the size of the histidine-heme loop increases, in accord with the Jacobson-Stockmayer equation. However, we observe that the His27-heme loop is both more stable than expected from the Jacobson-Stockmayer relationship and breaks more slowly than expected. These results show that the sequence near His27, which is in the reverse turn between helices 2 and 3 of UBA(2), is prone to persistent interactions in the denatured state. Therefore, consistent with our results for cytochrome c', this reverse turn sequence may help to establish the topology of this fold by biasing the conformational distribution of the denatured state.

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118–152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φeq value), which provides ...

The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay that can distinguish protein conformational changes close to and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs. A similar reversible structural transition of the active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to that for phLDH was accompanied by a larger contribution of entropy to ΔG, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine N-oxide (TMAO), increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO causes cgLDH to adopt catalytic parameters like those of phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme that have varied catalytic parameters (i.e., binding and catalytic proclivity) and whose population profiles are temperature-dependent and influenced by osmolytes interconvert among themselves. Our results also show that adaptation can be achieved by means other than gene mutations and complements the synchronic evolution of the cellular milieu.

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5–18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ 1/2 I2 ⇄ 1/3 I3 ⇄ 1/4 I4) including three new oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible...

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Diacylglycerol kinase ε (DGKε) catalyzes the phosphorylation of diacylglycerol, producing phosphatidic acid. DGKε demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the enrichment of particular acyl chains within the lipids of the phosphatidylinositol cycle. Phosphatidylinositol is highly enriched with 1-stearoyl-2-arachidonoyl, which is important for maintaining cellular health. Dysregulation of DGKε perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington's disease, and heart disease. Recessive loss-of-function mutations in the DGKε gene cause atypical hemolytic uremic syndrome. Because DGKε has never been purified, little is known about its molecular properties. We expressed human DGKε and a truncated version lacking the first 40 residues (DGKεΔ40) and purified both proteins to near homogeneity using nickel affinity chromatography. Kinase activity measurements showed that both purified constructs retained their acyl chain specificity for diacylglycerol with an activity level comparable to that of N-terminally FLAG epitope-tagged forms of these proteins expressed in COS7 cells. Both constructs lost activity upon being stored, particularly upon freezing and thawing, which was minimized by the addition of glycerol. Circular dichroism revealed that DGKε and DGKεΔ40 both contain significant amounts of α-helical and β structure and exhibit biphasic thermal denaturations. The loss of secondary structure upon heating was irreversible for both constructs, with relatively little effect of added dioleoylphosphatidylcholine. The addition of 50% glycerol stabilized both constructs and facilitated refolding of their secondary structures after heating. This is the first successful purification and characterization of DGKε's enzymatic and conformational properties. The purification of DGKε permits detailed analyses of this unique enzyme and will improve our understanding of DGKε-related diseases.

The structural basis of pathways of misfolding of a cellular prion (PrPC) into the toxic scrapie form (PrPSC) and identification of possible intermediates (e.g., PrP*) still eludes us. In this work, we have used a cumulative ∼65 μs of replica exchange molecular dynamics simulation data to construct the conformational free energy landscapes and capture the structural and thermodynamic characteristics associated with various stages of the thermal denaturation process in human prion protein. The temperature-dependent free energy surfaces consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds, thus rendering the protein prone to misfolding. We have been able to identify metastable conformational states with high β-content (∼30–40%) and low α-content (∼10–20%) that might be precursors of PrPSC oligomer formation. These conformations also involve participation of the unstructured N-terminal domain, and its role in misfolding has been investigated. All the misfolded or partial...

Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.

Protease inhibitors have the highest potency among antiviral therapies against HIV-1 infections, yet the virus can evolve resistance. Darunavir (DRV), currently the most potent Food and Drug Administration-approved protease inhibitor, retains potency against single-site mutations. However, complex combinations of mutations can confer resistance to DRV. While the interdependence between mutations within HIV-1 protease is key for inhibitor potency, the molecular mechanisms that underlie this control remain largely unknown. In this study, we investigated the interdependence between the L89V and L90M mutations and their effects on DRV binding. These two mutations have been reported to be positively correlated with one another in HIV-1 patient-derived protease isolates, with the presence of one mutation making the probability of the occurrence of the second mutation more likely. The focus of our investigation is a patient-derived isolate, with 24 mutations that we call "KY"; this variant includes the L89V and L90M mutations. Three additional KY variants with back-mutations, KY(V89L), KY(M90L), and the KY(V89L/M90L) double mutation, were used to experimentally assess the individual and combined effects of these mutations on DRV inhibition and substrate processing. The enzymatic assays revealed that the KY(V89L) variant, with methionine at residue 90, is highly resistant, but its catalytic function is compromised. When a leucine to valine mutation at residue 89 is present simultaneously with the L90M mutation, a rescue of catalytic efficiency is observed. Molecular dynamics simulations of these DRV-bound protease variants reveal how the L90M mutation induces structural changes throughout the enzyme that undermine the binding interactions.

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins.

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Since SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin (HPN). SERPINB12 may function as an endogenous inhibitor of these peptidases.

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71–85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (pKtrigger). A second deprotonation stabilizes a pentacoordinate heme form (pKa2). In this study, site-directed variants at the 41 and 48 positions in the 40–57 Ω-loop and at the 81 and 83 positions in the 71–85 Ω-loop reveal that conformational transitions in the 71–85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40–57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and...

There is considerable evidence that long-range interactions stabilize residual protein structure under denaturing conditions. However, evaluation of the effect of a specific contact on structure in the denatured state has been difficult. Iso-1-cytochrome c variants with a Lys54→His mutation form a particularly stable His-heme loop in the denatured state, suggestive of loop-induced residual structure. We have used multidimensional NMR methods to assign 1H and 15N backbone amide and 13C backbone and side chain chemical shifts in the denatured state of iso-1-cytochrome c carrying the Lys54→His mutation in 3 and 6 M guanidine hydrochloride and at both pH 6.4, where the His54-heme loop is formed, and pH 3.6, where the His54-heme loop is broken. Using the secondary structure propensity score, with the 6 M guanidine hydrochloride chemical shift data as a random coil reference state for data collected in 3 M guanidine hydrochloride, we found residual helical structure in the denatured state for the 60’s helix and the C-terminal helix, but not in the N-terminal helix in both the presence and absence of the His54-heme loop. Non-native helical structure is observed in two regions that form Ω-loops in the native state. There is more residual helical structure in the C-terminal helix at pH 6.4 when the loop is formed. Loop formation also appears to stabilize helical structure near His54, consistent with induction of helical structure observed when His-heme bonds form in heme-peptide model systems. The results are discussed in the context of the folding mechanism of cytochrome c.

A protein's stability may range from nonexistent, as in the case of intrinsically disordered proteins, to very high, as indicated by a protein's resistance to degradation, even under relatively harsh conditions. The stability of this latter group is usually under kinetic control because of a high activation energy for unfolding that virtually traps the protein in a specific conformation, thereby conferring resistance to proteolytic degradation and misfolding aggregation. The usual outcome of kinetic stability is a longer protein half-life. Thus, the protective role of protein kinetic stability is often appreciated, but relatively little is known about the extent of biological roles related to this property. In this Perspective, we will discuss several known or putative biological roles of protein kinetic stability, including protection from stressors to avoid aggregation or premature degradation, achieving long-term phenotypic change, and regulating cellular processes by controlling the trigger and timing of molecular motion. The picture that emerges from this analysis is that protein kinetic stability is involved in a myriad of known and yet to be discovered biological functions via its ability to confer degradation resistance and control the timing, extent, and permanency of molecular motion.

The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.

PagP is an eight-stranded transmembrane β-barrel enzyme indispensable for lipid A palmitoylation in Gram-negative bacteria. The severity of infection by pathogens, including Salmonella, Legionella, and Bordetella, and resistance to antimicrobial peptides, relies on lipid A remodeling by PagP, rendering PagP a sought-after drug target. Despite a conserved sequence, more robust palmitoylation of lipid A is observed in Salmonella typhimurium compared to Escherichia coli, a possible consequence of the differential regulation of PagP expression and/or specific activity. Work here identifies molecular signatures that demarcate thermodynamic stability and variances in catalytic efficiency between S. typhimurium (PagP-St) and E. coli (PagP-Ec) transmembrane PagP barrel variants. We demonstrate that Salmonella PagP displays a 2-fold destabilization of the barrel, while achieving 15-20 magnitude higher lipase efficiency, through subtle alterations of lipid-facing residues distal from the active site. We find that catalytic properties of these homologues are retained across different lipid environments such as micelles, vesicles, and natural extracts. By comparing thermodynamic stability with activity of selectively designed mutants, we conclude that activity-stability trade-offs can be influenced by factors secluded from the catalytic region. Our results provide a compelling correlation of the primary protein structure with enzymatic activity, barrel thermodynamic stability, and scaffold plasticity. Our analysis can open avenues for the development of potent pharmaceuticals against salmonellosis.

The structural characterization of low populated states of proteins with accuracy comparable to that achievable for native states is important for understanding the mechanisms of protein folding and function, as well as misfolding and aggregation. Because of the transient nature of these low populated states, they are seldom detected directly under conditions that favor folding. The activation domain of human procarboxypeptidase A2 (ADA2h) is an α/β-protein that forms amyloid fibrils at low pH, presumably initiated from a denatured state with a considerable amount of residual structure. Here we used Carr-Parcell-Meiboom-Gill relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the denatured state of the ADA2h I71V mutant under conditions that favor folding. Under these conditions, the lifetime of the denatured state of I71V ADA2h is on the order of milliseconds and its population is approximately several percent, which makes this mutant amenable to studies by CPMG RD methods. The nearly complete set of CPMG RD-derived backbone (15)N, (13)C, and (1)H NMR chemical shifts in the I71V ADA2h denatured state reveals that it retains a significant fraction (up to 50-60%) of nativelike α-helical structure, while the regions encompassing native β-strands are structured to a much lesser extent. The nativelike α-helical structure of the denatured state can bring together hydrophobic residues on the same sides of α-helices, making them available for intra- or intermolecular interactions. CPMG RD data analysis thus allowed a detailed structural characterization of the ADA2h denatured state under folding conditions not previously achieved for this protein.

The effects of a 10.5 M solution of aqueous urea on Helicobacter pylori urease were investigated over the course of a 500 ns molecular dynamics (MD) simulation. The enzyme was solvated by 25321 water molecules, and additionally, 4788 urea molecules were added to the solution. Although concentrated urea solutions are known laboratory denaturants, the protein secondary structure is retained throughout the simulation largely because of the short simulation time (urea denaturation occurs on the millisecond time scale). The relatively constant solvent accessible surface area over the last 400 ns of the simulation further confirms the overall lack of denaturation. The wide-open flap state observed previously in Klebsiella areogenes urease [Roberts, B. P., et al. (2012) J. Am. Chem. Soc. 134, 9934] and H. pylori [Minkara, M. S., et al. (2014) J. Chem. Theory Comput. 10, 1852-1862] was also identified in this aqueous urea simulation. Over the course of the trajectory, we were able to observe urea molecules entering the active site in proportions related to the extent of opening of the active site-covering flap. Furthermore, urea molecules were observed to approach the pentacoordinate Ni(2+) ion in position to bind in a manner consistent with the proposed initial coordination step of the hydrolysis mechanism. We also observed a specific and unique pattern in the regions of the protein with a high root-mean-square fluctuation (rmsf). The high-rmsf regions in the β-chain form a horseshoelike arrangement surrounding the active site-covering flap on the surface of the protein. We hypothesize that the function of these regions is to both attract and shuttle urea toward the loop of the active site-covering flap before entry into the cavity. Indeed, urea is observed to interact with these regions for extended periods of simulation time before active site ingress.

The reversible thermal denaturation of apo α-lactalbumin and lysozyme was monitored via measurement of changes in absorbance and ellipticity in the presence of varying concentrations of seven mono- and oligosaccharides: glucose, galactose, fructose, sucrose, trehalose, raffinose, and stachyose. The temperature dependence of the unfolding curves was quantitatively accounted for by a two-state model, according to which the free energy of unfolding is increased by an amount that is independent of temperature and depends linearly upon the concentration of added saccharide. The increment of added unfolding free energy per mole of added saccharide was found to depend approximately linearly upon the extent of oligomerization of the saccharide. The relative strength of stabilization of different saccharide oligomers could be accounted for by a simplified statistical-thermodynamic model attributing the stabilization effect to volume exclusion deriving from steric repulsion between protein and saccharide molecules.

Human apolipoprotein A-1 (Apo A-1) was used as a model protein to compare experimental methods and theoretical models for protein unfolding. Thermal unfolding was investigated in aqueous buffer, in β-octylglucoside solution, and with phospholipid bilayer vesicles. The α-helix content of Apo A-1 increased from 50% in aqueous buffer to 75% in the presence of lipid vesicles, but remained constant in solutions of β-octyl glucoside. Differential scanning calorimetry (DSC) measured the thermodynamic properties of the unfolding process and was our reference method. The increased heat capacity of the unfolded protein made an important contribution to the total enthalpy of unfolding. The structural properties of Apo A-1 were studied with circular dichroism (CD) spectroscopy. The CD-recorded unfolding transitions were broader than the corresponding DSC transitions and were shifted toward higher temperatures. DSC and CD data were analyzed with the two-state model and the Zimm-Bragg theory. The two-state model assumes just two species in solution, native (N) and unfolded (U) Apo A-1. However, Apo A-1 unfolding is a highly cooperative event with helical amino acid residues unfolding and refolding rapidly. For such a sequential process, the Zimm-Bragg theory provides an alternative and physically more realistic model. The Zimm-Bragg theory allowed perfect simulations of the DSC and CD experiments. In contrast, incorrect thermodynamic results were obtained with the two-state model. The Zimm-Bragg theory also provided a physically well-defined analysis of the cooperativity of the folding ⇄ unfolding equilibrium. The cooperative unfolding of Apo A-1 increased upon addition of lipids and decreased in detergent solution.

The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7–10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the Ki for inorganic phosphate (product inhibition) and the KM for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and...

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo HX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chemical HX rate. In this work, we evaluate several different methods of obtaining an accurate model for the chemical HX rate suitable for millisecond hydrogen exchange mass spectrometry (HX-MS) analysis of disordered proteins: (1) calculations using the method of Englander [Bai, Y., et al. (1993) Proteins 17, 75-86], (2) measurement of HX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of HX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors and the CREB binding protein as the model IDPs, we show that the Englander method has slight inaccuracies that lead to underestimation of the chemical exchange rate. Second, HX-MS measurements of model peptides show that HX rates are changed dramatically by high concentrations of the denaturant. Third, we find that measurements of HX by reference peptides from the proteins of interest provide the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond HX-MS.

Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), ligation of Met80 to the heme iron is critical for the protein's electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron.

Engineered proteins provide clinically and industrially impactful molecules and utility within fundamental research. Yet, inefficiencies in discovering lead variants with new desired function, while maintaining stability, hinder progress. Improved function, which can result from a few strategic mutations, is fundamentally separate from discovering novel function, which often requires large leaps in sequence space. While a highly diverse combinatorial library covering immense sequence space would empower protein discovery, the ability to sample only a minor subset of sequence space and the typical destabilization of random mutations preclude this strategy. A balance must be reached. At library scale, compounding several destabilizing mutations renders many variants unable to properly fold and devoid of function. Broadly searching sequence space while reducing destabilization may enhance evolution. We exemplify this balance with affibody, a three-helix bundle protein scaffold. Using natural ligand datasets, stability and structural computations, and deep sequencing thousands of binding variants, a protein library was designed on a sitewise basis with a gradient of mutational levels across 29% of the protein. In direct competition of biased and uniform libraries, both with 1×109 variants, for discovery of 6×104 ligands (5×103 clusters) toward seven targets, biased amino acid frequency increased ligand discovery 13±3-fold. Evolutionarily favorable amino acids, both globally and site-specifically, are further elucidated. The sitewise amino acid bias aids evolutionary discovery by reducing mutant destabilization as evidenced by 15°C higher midpoint of denaturation relative to unbiased mutants (62±4°C vs. 47±11°C, p