Acid-denatured State Research Articles

Acid-Induced Unfolding of Champedak Galactose-Binding Lectin.

Acid denaturation of champedak galactose-binding (CGB) lectin was studied in the pH range, 7.0-1.0 using intrinsic fluorescence and ANS fluorescence measurements. The lectin remained stable up to pH 5.0 and showed local disordering in the vicinity of the protein fluorophores within the pH range, 5.0-3.5. Decrease in the pH from pH 3.5 to pH 2.5 led to structural transition, marked by the decrease in the intrinsic fluorescence and increase in the ANS fluorescence signals. This can be ascribed to the dissociation of the tetrameric lectin into monomeric forms. Further decrease in the pH up to pH 1.5 produced another transition, which specified the unfolding of monomers as reflected from the decrease in both intrinsic fluorescence and ANS fluorescence signals. Characterization of the conformational states obtained at pH 7.0, pH 2.5 and pH 1.5 based on intrinsic and ANS fluorescence spectra, gel chromatographic behavior and thermal denaturation confirmed the existence of folded monomeric forms at pH 2.5 and unfolded states at pH 1.5. However, the aciddenatured state of CGB lectin at pH 1.5 retained significant residual structure, as evident from the greater loss of both secondary and tertiary structures in the presence of 6 M guanidine hydrochloride at low pH values. Anion-induced refolding below pH 1.5 was also seen using ANS fluorescence measurements.

Solution Behavior and Interaction of Pepsin with Carnitine Based Cationic Surfactant: Fluorescence, Circular Dichroism, and Calorimetric Studies.

The present work reports the pH-induced conformational changes of pepsin in solution at room temperature. The conformational change makes the protein surface active. The protein was found to be present in the partially denatured state at pH 8 as well as at pH 2. The fluorescence probe and circular dichroism (CD) spectra suggested that the most stable state of pepsin exists at pH 5. The binding affinities of pepsin in its native and denatured states for a D,L-carnitine-based cationic surfactant (3-hexadecylcarbamoyl-2-hydroxypropyl)trimethylammonium chloride (C16-CAR) were examined at very low concentrations of the surfactant. The thermodynamics of the binding processes were investigated by use of isothermal titration calorimetry. The results were compared with those of (3-hexadecylcarbamoylpropyl)trimethylammonium chloride (C16-PTAC), which is structurally similar to C16-CAR, but without the secondary -OH functionality near the headgroup. None of the surfactants were observed to undergo binding with pepsin at pH 2, in which it exists in the acid-denatured state. However, both of the surfactants were found to spontaneously bind to the most stable state at pH 5, the partially denatured state at pH 8, and the alkaline denatured state at pH 11. Despite the difference in the headgroup structure, both of the surfactants bind to the same warfarin binding site. Interestingly, the driving force for binding of C16-CAR was found to be different from that of C16-PTC at pH ≥ 5. The steric interaction of the headgroup in C16-CAR was observed to have a significant effect on the binding process.

Statistical mechanics of the denatured state of a protein using replica-averaged metadynamics.

The characterization of denatured states of proteins is challenging because the lack of permanent structure in these states makes it difficult to apply to them standard methods of structural biology. In this work we use all-atom replica-averaged metadynamics (RAM) simulations with NMR chemical shift restraints to determine an ensemble of structures representing an acid-denatured state of the 86-residue protein ACBP. This approach has enabled us to reach convergence in the free energy landscape calculations, obtaining an ensemble of structures in relatively accurate agreement with independent experimental data used for validation. By observing at atomistic resolution the transient formation of native and non-native structures in this acid-denatured state of ACBP, we rationalize the effects of single-point mutations on the folding rate, stability, and transition-state structures of this protein, thus characterizing the role of the unfolded state in determining the folding process.

Halogenol- versus alkanol-induced structural transitions of acid-denatured glucoamylase: Characterization of alcohol-induced states

Different probes such as far- and near-UV CD spectral signals, ANS binding, Trp fluorescence and three-dimensional fluorescence were used to study halogenol- versus alkanol-induced conformational transitions in the acid-denatured state (pH 1.0) of Aspergillus niger glucoamylase (GA). These alcohols showed significant retrieval of the protein structure, inducing both secondary and tertiary structural changes, as evident from the increase in the α-helix and decrease in ANS binding, respectively. However, halogenols were found more competent than alkanols, requiring lesser alcohol concentration to induce similar spectral change. The effectiveness of these alcohols showed the order: HFIP>TFE>2-chloroethanol for halogenols while tert-butanol>1-propanol>2-propanol>ethanol>methanol for alkanols. Both Trp fluorescence and near-UV CD spectra showed anomalous pattern, though the order of effectiveness remained the same as found with far-UV CD spectra and ANS fluorescence. Three-dimensional fluorescence results of the acid-denatured state (pH 1.0) of GA in the presence of 5.5M tert-butanol agreed well with the data obtained from far-UV CD and Trp fluorescence. All these results suggested the formation of partially folded states of GA obtained in the presence of these alcohols, being more effective with halogenols than alkanols.

Generating Accurate Contact Maps of Transient Long-Range Interactions in Intrinsically Disordered Proteins by Paramagnetic Relaxation Enhancement

Generating Accurate Contact Maps of Transient Long-Range Interactions in Intrinsically Disordered Proteins by Paramagnetic Relaxation Enhancement

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β(2)-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH7.5 to pH2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH2.0 to pH7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5-6 s(-1), while the molecule with the Pro32 trans isomer refolds more slowly (pH7.5 and 25°C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001-0.002 s(-1)) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7-9% under a more physiological condition (pH7.5 and 37°C).

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

The acid transition of β(2)-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH4 (25°C). The pH4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK(a) value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state→MG→burst-phase intermediate→native state.

The Monomer–Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE222nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE222nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in Ksv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100°C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.

Thermal and acid denaturation of bovine lens α‐crystallin

The chaperone-like protein α-crystallin is a ∼35 subunit hetero-oligomer consisting of αA and αB subunits in a 3:1 molar ratio and has the function of maintaining eye lens transparency. We studied the thermal denaturation of α-crystallin by differential scanning calorimetry (DSC), circular dichroism (CD), and dynamic light scattering (DLS) as a function of pH. Our results show that between pH 7 and 10 the protein undergoes a reversible thermal transition. However, the thermodynamic parameters obtained by DSC are inconsistent with the complete denaturation of an oligomeric protein of the size of α-crystallin. Accordingly, the CD data suggest the presence of extensive residual secondary structure above the transition temperature. Within the pH range from 4 to 7 the increased aggregation propensity around the isoelectric point (pI ∼ 6) precludes observation of a thermal transition. As pH decreases below 4 the protein undergoes a substantial unfolding. The secondary structure content of the acid-denatured state shows little sensitivity to heating. We propose that the thermal transition above pH 7 and the acid-induced transition at ambient temperature result in predominant denaturation of the αB subunit. Although the extent of denaturation of the αA subunit cannot be estimated from the current data, the existence of a native-like conformation is suggested by the preserved association of the subunits and the chaperone-like activity. A key difference between the thermal and the acid denaturation is that the latter is accompanied by dissociation of αB subunits from the remaining αA-oligomer, as supported by DLS studies.

Carboxyl p Ka Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the p K a values of all the carboxylic residues in this pH range. We employed 13C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring p K a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered p K a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed p K a, and, using thermodynamic cycles, we could calculate their p K as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured 13C α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.

Modulating Native-like Residual Structure in the Fully Denatured State of Photoactive Yellow Protein Affects Its Refolding

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

Partially Folded States of Mutant Ubiquitin in Mild Denaturing Conditions

Conformational change of ubiquitin variant with valine to alanine mutation at sequence position 26 was studied by varying solvent pH. Fluorescence emission spectra indicated that this variant ubiquitin has some residual structures in acidic and basic solution as compared to denaturant-induced unfolded state. Far-UV circular dichroic spectra indicated that the base-denatured state had more secondary structure than the acid-denatured state. Near-UV circular dichroic spectra indicated that the aromatic side-chains were in the relatively more rigid environment in the base-denatured state than those in the acid-denatured state. Although it appears that the more tertiary structure present in the base-denatured state, refolding reactions measured by stopped-flow fluorescence device suggest that both the acid- and base-denatured states occur before the major folding transition state. The acid- and base-denatured states are considered to reflect the early event of protein folding process.

Competition between Intramolecular and Intermolecular Interactions in an Amyloid-Forming Protein

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human β2-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded β2-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.

A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0

Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 degrees C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 degrees C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 degrees C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.

Chromophore Isomerization Has Large Effects On The Residual Structure Of The Fully Unfolded State Of The Blue Light Receptor Photoactive Yellow Protein

Protein folding occurs between a well-defined fully folded native state and a structurally much less studied fully unfolded state. We use denaturant m values and changes in heat capacity ΔCp to probe folding transitions in photoactive yellow protein (PYP). PYP is a bacterial photoreceptor that exhibits rhodopsin-like photochemistry based on the trans to cis photoisomeriation of its covalently attached p-coumaric acid (pCA) chromophore. We report strong effects of the isomerization state of the pCA on the residual structure of the “fully unfolded” state of PYP by comparing the unfolding of two partially unfolded states of PYP: the acid-denatured state pBdark, which contains trans-pCA, and the partially unfolded pB photocycle intermediate, which contains cis-pCA.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and 15N relaxation data ( R 1, R 2, NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

Green fluorescent protein (GFP) is a large protein with a complex eleven-stranded beta-barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of HD exchange measurement and nuclear magnetic resonance (NMR) techniques to probe the energy landscape for folding of GFP in further detail. HD exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. HD exchange experiments at high pH establish that there is an extremely slow-exchanging superstable core of amide protons in GFP that are clustered and located in beta-strands 1, 2, 4, 5, and 6. These residues form part of a mini-beta-sheet which we propose constitutes a folding nucleus. Using a pulsed-labeling strategy, the acid-denatured state has been investigated and the residual structure observed in earlier studies shown to locate to beta-strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain.

Model-independent interpretation of NMR relaxation data for unfolded proteins: the acid-denatured state of ACBP

We have investigated the acid-unfolded state of acyl-coenzyme A binding protein (ACBP) using 15N laboratory frame nuclear magnetic resonance (NMR) relaxation experiments at three magnetic field strengths. The data have been analyzed using standard model-free fitting and models involving distribution of correlation times. In particular, a model-independent method of analysis that does not assume any analytical form for the correlation time distribution is proposed. This method explains correlations between model-free parameters and the analytical distribution parameters found by other authors. The analysis also shows that the relaxation data are consistent with and complementary to information obtained from other parameters, especially secondary chemical shifts and residual dipolar couplings, and strengthens the conclusions of previous observations that three out of the four regions that form helices in the native structure appear to contain residual secondary structure also in the acid-denatured state.

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26-32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.