Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.     

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant

The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.     

The fluorescence detected guanidine hydrochloride equilibrium denaturation of wild-type staphylococcal nuclease does not fit a three-state unfolding model

A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.     

Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensis delta-endotoxins.

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.     

Existence of common antigenic sites in tropomyosins.

1. Tropomyosins were extracted from vertebrate and invertebrate muscles, and their immunolo;ical characteristics were compared using antisera against tropomyosins from chicken skeletal and cardiac muscles. 2. Antigenic sites common to those of chicken skeletal muscle tropomyosin were found in all the tropomyosins tested, although the reactions of these common antigenic sites in an immunodiffusion test were weak in tropomyosins from phylogenetically distant animals. 3. An immunological difference was found between alpha-tropomyosins from chicken cardiac muscle and rabbit cardiac muscle. Thus they had specific antigenic sites in addition to the common ones. 4. A component was found in a 1 M KCL extract of Tetrahymena pyriformis which reacted with antiserum against chicken skeletal muscle tropomyosin.     

Probing conformational changes in orphan nuclear receptor: the NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.     

Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas

Thiol proteinase inhibitors are crucial to proper functioning of all living tissues consequent to their cathepsin regulatory and myriad important biologic properties. Equilibrium denaturation of dimeric goat pancreas thiol proteinase inhibitor (PTPI), a cystatin superfamily variant has been studied by monitoring changes in the protein's spectroscopic and functional characteristics. Denaturation of PTPI in guanidine hydrochloride and urea resulted in altered intrinsic fluorescence emission spectrum, diminished negative circular dichroism, and loss of its papain inhibitory potential. Native like spectroscopic properties and inhibitory activity are only partially restored when denaturant is diluted from guanidine hydrochloride unfolded samples demonstrating that process is partially reversible. Coincidence of transition curves and dependence of transition midpoint (3.2M) on protein concentration in guanidine hydrochloride‐induced denaturation are consistent with a two‐state model involving a native like dimer and denatured monomer. On the contrary, urea‐induced unfolding of PTPI is a multiphasic process with indiscernible intermediates. The studies demonstrate that functional conformation and stability are governed by both ionic and hydrophobic interactions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 708–717, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.     

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.     

Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.     

Synthesis and structural stability of helichrome as an artificial hemeproteins

A detailed procedure is described for the synthesis of helichrome, which is the first successful example of polypeptide-based artificial hemeprotein. The segment synthesis-condensation approach used for the assembly of small proteins has proven to be extremely useful for protein mimetics as well. The final deprotection was performed using the TMSOTf-thioanisole method instead of the less-convenient hydrogen fluoride method. The unfolding transition of the alpha-helical conformation of helichrome induced by guanidine hydrochloride was studied to understand the stability and dynamics of the folded structure. The resulting parameters (C0.5 = 5.2 M and delta GH2O = -4.4 kcal mol-1) characterizing helichrome denaturation were comparable to that of native globular proteins.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unfolding behavior of human alpha 1-acid glycoprotein is compatible with a loosely folded region in its polypeptide chain

The unfolding of human plasma alpha 1-acid glycoprotein (AGP) induced by heat or guanidine hydrochloride was studied under equilibrium conditions. In thermal unfolding, an intermediate state was detected by the appearance of unusual positive difference absorption bands in the 287-295-nm region, which occurred at lower temperatures than the common denaturation bands at 284 and 291 nm. The formation of this intermediate species apparently involves a local conformational change that perturbs the environment of tryptophyl residues, without affecting the secondary structure of the protein as judged from circular dichroism spectra. On the other hand, denaturation of the glycoprotein induced by guanidine hydrochloride seemed to follow a two-state model with no evidence of any intermediate species; however, the analysis of the transition curve indicated that the change in the accessibility to solvent of amino acid residues of AGP upon unfolding is significantly lower than those observed for other proteins. According to these results, it is proposed that part of the polypeptide chain in native AGP, namely, that from residue 122 to the C-terminus, may be "loosely" folded.     

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.     

New insights into the allergenicity of tropomyosin: a bioinformatics approach

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133–135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.