The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding

Antichymotrypsin, a member of the serpin superfamily, has been shown to form inactive polymers in vivo, leading to chronic obstructive pulmonary disease. At present, however, the molecular determinants underlying the polymerization transition are unclear. Within a serpin, the breach position is implicated in conformational change, as it is the first point of contact for the reactive center loop and the body of the molecule. W194, situated within the breach, represents one of the most highly conserved residues within the serpin architecture. Using a range of equilibrium and kinetic experiments, the contribution of W194 to proteinase inhibition, stability and polymerization was studied for antichymotrypsin. Replacement of W194 with phenylalanine resulted in a fully active inhibitor that was destabilized relative to the wild-type protein. The aggregation kinetics were significantly altered; wild-type antichymotrypsin exhibits a lag phase followed by chain elongation. The loss of W194 almost entirely removed the lag phase and accelerated the elongation phase. On the basis of our data, we propose that one of the main roles of W194 in antichymotrypsin is in preventing polymerization.     

Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin

Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HgammaD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus.     

Escherichia coli OmpA retains a folded structure in the presence of sodium dodecyl sulfate due to a high kinetic barrier to unfolding.

Escherichia coli OmpA can be solubilized by sodium dodecyl sulfate (SDS) in its folded structure, and it unfolds upon heating. Although the heat-denatured OmpA remains unfolded after lowering the temperature, the addition of a non-ionic surfactant, octyl glucoside results in refolding of unfolded OmpA. In the present study, we investigated the refolding kinetics of OmpA in a mixed surfactant system of SDS and octyl glucoside using far- and near-UV circular dichroism and fluorescence spectroscopies. We found four kinetic phases in the refolding reaction, which logarithmically depended on the weight fraction of octyl glucoside. We also examined the unfolding kinetics of OmpA upon heating in the presence of SDS by temperature jump experiments. A comparison of the rate constants for the refolding and the unfolding reactions in SDS-only solution at 30 degrees C revealed that the folded form of OmpA in SDS solution is less stable than the unfolding form, and that the unfolding is virtually unobservable near room temperature due to a high kinetic barrier.     

ClpX-mediated remodeling of mu transpososomes: selective unfolding of subunits destabilizes the entire complex.

E. coli ClpX, a member of the Clp/Hsp100 family of ATPases, remodels multicomponent complexes and facilitates ATP-dependent degradation. Here, we analyze the mechanism by which ClpX destabilizes the exceedingly stable Mu transpososome, a natural substrate for remodeling rather than degradation. We find that ClpX has the capacity to globally unfold transposase monomers, the building blocks of the transpososome. A biochemical probe for protein unfolding reveals that ClpX also unfolds MuA subunits during remodeling reactions, but that not all subunits have their structure extensively modified. In fact, direct recognition and unfolding of a single transposase subunit are sufficient for ClpX to destabilize the entire transpososome. Thus, the ability of ClpX to unfold proteins is sufficient to explain its role in both complex destabilization and ATP-dependent proteolysis.     

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.     

Diffusional barrier in the unfolding of a small protein

To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.     

Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics

Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded beta-lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of epsilon-amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by approximately 3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5 degrees C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications.     

Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.     

Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T_m) by 6–7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) ⇄ I_T(±MgATP) → A_T → (A_T)_n. The equilibrium unfolding to intermediate, I_T, is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A_T, for which aggregation to (A_T)_n and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A_T.     

Free energy barrier estimation of unfolding the alpha-helical surfactant-associated polypeptide C

Molecular dynamics simulations were conducted to estimate the free energy barrier of unfolding surfactant-associated polypeptide C (SP-C) from an alpha-helical conformation. Experimental studies indicate that while the helical fold of SP-C is thermodynamically stable in phospholipid micelles, it is metastable in a mixed organic solvent of CHCl3/CH3OH/0.1 M HCl at 32:64:5 (v/v/v), in which it undergoes an irreversible transformation to an insoluble aggregate that contains beta-sheet. On the basis of experimental observations, the free energy barrier was estimated to be approximately 100 kJ/mole by applying Eyring's transition state theory to the experimental rate of unfolding [Protein Sci 1998;7:2533-2540]. These studies prompted us to carry out simulations to investigate the unwinding process of two helical turns encompassing residues 25-32 in water and in methanol. The results give an upper bound estimation for the free energy barrier of unfolding of SP-C of approximately 20 kJ/mole. The results suggest a need to reconsider the applicability of a single-mode activated process theory to protein unfolding.     

Mutant copper-zinc superoxide dismutase binds to and destabilizes human low molecular weight neurofilament mRNA

The mechanism by which mutated copper-zinc superoxide dismutase (SOD1) causes familial amyotrophic lateral sclerosis is believed to involve an adverse gain of function, independent of the physiological antioxidant enzymatic properties of SOD1. In this study, we have observed that mutant SOD1 (G41S, G85A, and G93A) but not the wild type significantly reduced the stability of the low molecular weight neurofilament mRNA in a dosage-dependent manner. We have also demonstrated that mutant SOD1 but not the wild type bound directly to the neurofilament mRNA 3'-untranslated region and that the binding was necessary to induce mRNA destabilization. These observations provide an explanation for a novel gain of function in which mutant SOD1 expression in motor neurons alters an intermediate filament protein expression.     

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Deamidation in human lens betaB2-crystallin destabilizes the dimer

Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. betaB2 is the most abundant beta-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. betaB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among beta-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type betaB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes betaB2 and may disrupt the function of betaB2 in the lens.     

Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.     

Equilibrium and kinetic studies of unfolding of homologous cytochromes c.

Extensive investigations of the unfolding equilibria and kinetics of oxidized and reduced cytochromes c are reported. It is found that all cytochromes c have similar unfolding free energies (deltaGD = 7 +/- 1 kcal/mol). Differences among species do not correlate in any way with the metabolic differences among species. The stabilization of cytochrome c on reduction is estimated at 1.1 kcal/mol. Stability differences among species are mirrored in their denaturation kinetics. For cytochrome c (III), the unfolding exhibits multiple phases. The rate constants for the two observable phases both change by a factor of 3 between horse cytochrome c (III) and cow cytochrome c (III). On reduction, all unfolding appears to occur in a single step. The rate of this unfolding still varies between species, however, the results can be accommodated to a sequential model, with some assumptions. The observations are consistent with chain reversal occurring at an early stage in the reaction and suggest that previously observed rapid processes may be ligand exchange processes.     

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.