Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH

The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.     

The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH.

The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 degrees C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is deltaG = 4.0 +/- 0.2 kcal x mol-1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7-9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5-4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 +/- 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.     

Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate.

The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV-sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, beta-mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interact directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation-competent prefolded conformation, which may readily lead to a correctly folded enzyme.     

Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH

A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfolded papain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features in the presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain was greater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a compact "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule-like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds.     

High-resolution structure of a partially folded insulin aggregation intermediate

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details—the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.     

Salt-induced refolding of myoglobin at acidic pH: molecular properties of a partly folded intermediate.

The molecular properties of the salt-induced partly folded acidic state of apomyoglobin as well as myoglobin were investigated by fluorescence and circular dichroism of the extrinsic fluorophore 1,8-anilinonaphthalenesulfonate. The occurrence of a fluctuating tertiary structure ("molten globule") at acidic pH in the presence of salt was suggested by the disappearance of the dichroic activity of the fluorophore bound to the partly folded protein. Moreover, the structure of the intermediate is not influenced by the presence of heme, thus suggesting that heme is not crucial in the early stage of myoglobin folding.     

Evidence for a partially folded intermediate in alpha-synuclein fibril formation

Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.     

Molten-globule like partially folded states of human serum albumin induced by fluoro and alkyl alcohols at low pH

Human serum albumin (HSA) exists in a molten-globule like state at low pH (pH 2.0). We studied the effects of trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) on the acid-denatured state of HSA by far-UV circular dichroism (CD), near-UV CD, tryptophan fluorescence, and 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. At pH 2.0, these alcohols induced the formation of alpha-helical structure as evident from the increase in mean residue ellipticity (MRE) value at 222 nm. On addition of different alcohols, HSA exhibited a transition from the acid-denatured state to the alpha-helical state and loss of ANS-binding sites reflected by the decrease in ANS fluorescence at 480 nm. However, the concentration range required to bring about the transition varied greatly among different alcohols. HFIP was found to have highest potential whereas methanol was least effective in inducing the transition. The order of effectiveness of alcohols was shown to be: HFIP > TFE > 2-chloroethanol > tert-butanol > isopropanol > ethanol > methanol as evident from the Cm values. The near-UV CD spectra and tryptophan fluorescence showed the differential effects of halogenated alcohols with those of alkanols. A comparison of the m values, showing the dependence of Delta GH on alcohol concentration, suggests that the helix stabilizing potential of different alcohols is due to the additive effect of different constituent groups present whereas remarkably higher potential of HFIP involves some other factor in addition to the contribution of constituent groups.     

Characterization of two partially unfolded intermediates of the molecular chaperone DnaK at low pH

In this study, the effect of pH on the conformation and the reactivity of the Escherichia coli Hsp70 molecular chaperone DnaK was investigated using spectroscopic and chemical assays. DnaK exhibits negligible binding of the hydrophobic dye 1-anilino-naphthalene-8-sulfonate (ANS) between pH 7 to 5.0, whereas appreciable binding occurs between pH 4.5 to 4.0. The binding of ANS to a protein is diagnostic of the presence of accessible ordered hydrophobic surfaces. Such hydrophobic surfaces are often displayed by partially folded protein intermediates such as molten globules. Nucleotide inhibits 70% of the ANS binding at pH 4.5 but none of the ANS binding at pH 4.0. Proteolysis of nucleotide-free DnaK at pH 4.5 with cathepsin D yields detectable fragments (masses > 20 kDa) of the C-terminal peptide-binding domain but none of the N-terminal ATPase domain, thus the ATPase domain is preferentially targeted for proteolysis. In contrast, proteolysis of nucleotide-free DnaK at pH 4.0 with cathepsin D cuts near the linker region, yielding both functional domains. Our interpretation of these data is that incubation of DnaK at pH 4.5 produces a partially unfolded form of the ATPase domain, in which secondary structure is mainly intact, but tertiary structure is reduced. Incubation of the protein at pH 4.0 produces an intermediate in which both functional domains have collapsed and possibly separated. Nucleotide inhibits the conformational change that occurs at pH 4.5 but not at 4.0.     

Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.     

Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.     

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

HX-ESI-MS and optical studies of the unfolding of thioredoxin indicate stabilization of a partially unfolded, aggregation-competent intermediate at low pH

Hydrogen exchange monitored by mass spectrometry (HX-MS), in conjunction with multiple optical probes, has been used to characterize the unfolding of thioredoxin. Equilibrium and kinetic studies have been carried out at pH 7 and 3. The HX-MS measurements are shown to be capable of distinguishing between native (N) and unfolded (U) protein molecules when both are present together, and their application in kinetic experiments allows the unfolding reaction to be delineated from the proline isomerization reaction to which it is coupled. At pH 7, equilibrium unfolding studies monitored by three optical probes, intrinsic fluorescence at 368 nm, ellipticity at 222 nm, and ellipticity at 270 nm, as well as by HX-MS, indicate that no intermediate is populated at pH 7, the unfolding reaction is slower than the proline isomerization reaction that follows it, and the three optical probes yield identical kinetics for unfolding, which occurs in a single kinetic phase. The fractional change in any of the three optical signals at any time of unfolding predicts the fraction of the molecules that have become U, as determined by HX-MS. Hence, unfolding at pH 7 appears to occur via a two-state N <==> U mechanism. In contrast at pH 3, HX-MS as well as optical measurements indicate that an unfolding intermediate is stabilized and hence accumulates in equilibrium with N and U, at concentrations of denaturant that define the transition zone of the equilibrium unfolding curve. The intermediate has lost the near-UV signal characteristic of N and possesses fewer amide hydrogen sites that are stable to exchange than does N. Kinetic experiments at pH 3, where unfolding is much faster than proline isomerization, show that more than one intermediate accumulates transiently during unfolding. Thus, the unfolding of thioredoxin occurs via an N <==> I <==> U mechanism, where I is a partially unfolded intermediate that is stabilized and hence populated at pH 3 but not at pH 7. It is shown that transient aggregation of this intermediate results in a deceleration of the kinetics of unfolding at high protein concentrations at pH 3 but not at pH 7.     

Characterization of NMR relaxation-active motions of a partially folded A-state analogue of ubiquitin

The dominant dynamics of a partially folded A-state analogue of ubiquitin that give rise to NMR 15N spin relaxation have been investigated using molecular dynamics (MD) computer simulations and reorientational quasiharmonic analysis. Starting from the X-ray structure of native ubiquitin with a protonation state corresponding to a low pH, the A-state analogue was generated by a MD simulation of a total length of 33 ns in a 60%/40% methanol/water mixture using a variable temperature scheme to control and speed up the structural transformation. The N-terminal half of the A-state analogue consists of loosely coupled native-like secondary structural elements, while the C-terminal half is mostly irregular in structure. Analysis of dipolar N-H backbone correlation functions reveals reorientational amplitudes and time-scale distributions that are comparable to those observed experimentally. Thus, the trajectory provides a realistic picture of a partially folded protein that can be used for gaining a better understanding of the various types of reorientational motions that are manifested in spin-relaxation parameters of partially folded systems. For this purpose, a reorientational quasiharmonic reorientational analysis was performed on the final 5 ns of the trajectory of the A-state analogue, and for comparison on a 5 ns trajectory of native ubiquitin. The largest amplitude reorientational modes show a markedly distinct behavior for the two states. While for native ubiquitin, such motions have a more local character involving loops and the C-terminal end of the polypeptide chain, the A-state analogue shows highly collective motions in the nanosecond time-scale range corresponding to larger-scale movements between different segments. Changes in reorientational backbone entropy between the A-state analogue and the native state of ubiquitin, which were computed from the reorientational quasiharmonic analyses, are found to depend significantly on motional correlation effects.     

Low versus high molecular weight poly(ethylene glycol)-induced states of stem bromelain at low pH: stabilization of molten globule and unfolded states

The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.     

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.     

Characterization of fluoroalcohols-induced intermediates of Mucor miehei lipase at low pH

We have previously characterized an acid-unfolded (U(A)) state of Mucor miehei lipase at pH 2. The effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) resulted in characterization of molten-globule (MG) like states with beta-sheet secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. alpha-Helical states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.     

The structure and dynamics of partially folded actin.

Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.