A partially unfolded state of equine beta-lactoglobulin at pH 8.7

The urea-induced unfolding transition of equine -lactoglobulin was studied at pH 8.7 using circular dichroism (CD), ultracentrifugation, and gel filtration chromatography. The unfolding transition curves showed that at least one intermediate accumulates at moderate concentrations of urea. Furthermore, analytical ultracentrifugation experiments indicated that the intermediate forms a dimer. Thus, the urea-induced unfolding transition was measured by CD at various protein concentrations and was analyzed by a model assuming the four conformational states (the native, intermediate, dimeric intermediate, and unfolded states). The characteristics of the intermediate are markedly different from those of the intermediate previously observed at pH 4.0 or 1.5. The intermediate at pH 8.7 does not show the intense far-ultraviolet CD suggestive of the nonnative -helix.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.     

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 conformation is induced in pectate lyase C by the addition of 8-anilino-1-naphthalenesulfonate (ANS)

Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectate lyase C (pelC) leads to a large increase in the fluorescence quantum yield near 480 nm. The conventional interpretation of such an observation is that the ANS is binding to a partially folded intermediate such as a molten globule. Far-ultraviolet circular dichroism demonstrates that the enhanced fluorescence results from the induction of a partially folded protein species that adopts a large fraction of native-like secondary structure on binding ANS. Thus, ANS does not act as a probe to detect a partially folded species, but induces such a species. Near-ultraviolet circular dichroism suggests that ANS is bound to the protein in a specific conformation. The mechanism of ANS binding and structure induction was probed. The interaction of acid-unfolded pelC with several ANS analogs was investigated. The results strongly indicate that the combined effects of hydrophobic and electrostatic interactions account for the relatively high binding affinity of ANS for acid-unfolded pelC. These results demonstrate the need for caution in interpreting enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.     

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.     

Molecular dynamics simulations of the native and partially folded states of ubiquitin: influence of methanol cosolvent, pH, and temperature on the protein structure and dynamics

A series of explicit-solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a partially folded state (A-state) that is known to exist at low pH in methanol-water mixtures. The main results of the simulations are: (1) The ubiquitin native structure is remarkably stable at neutral pH in water; (2) the addition of the methanol cosolvent enhances the stability of the secondary structure but weakens tertiary interactions within the protein; (3) this influence of methanol on the protein structure is enhanced at low pH, while the effect of lowering the pH in pure water is limited; and (4) the A-state of ubiquitin can be described as a set of relatively rigid secondary structure elements (a native-like beta-sheet and native-like alpha-helix plus two nonnative alpha-helices) connected by flexible linkers.     

Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates.

Interaction with 8-anilino-1-naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates. Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and alpha-lactalbumin have been compared with conventional single-mix experiments and large discrepancies observed. When ANS was present throughout refolding, time-dependent changes attributed to the formation or reorganization of protein-ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding. The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states.     

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat,trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.     

pH-dependent stability of the human alpha-lactalbumin molten globule state: contrasting roles of the 6 - 120 disulfide and the beta-subdomain at low and neutral pH

Many proteins are capable of populating partially folded states known as molten globule states. Alpha-lactalbumin forms a molten globule under a range of conditions including low pH (the A-state) and at neutral pH in the absence of Ca(2+) with modest amounts of denaturant. The A-state is the most thoroughly characterized and thought to mimic a kinetic intermediate populated during refolding at neutral pH. We demonstrate that the properties and interactions that stabilize the A-state and the pH 7 molten globule of human alpha-lactalbumin differ. The unfolding of the wild-type protein is compared to the unfolding of a variant that lacks the 6 - 120 disulfide bond and to an autonomously folded peptide construct that we have previously shown represents the minimum core structure of the A-state of human alpha-lactalbumin. Studies conducted at pH 2 and 7 show that the disulfide makes little contribution to the stability of the molten globule at pH 7 but is important at pH 2. In contrast, the beta-subdomain of the protein is less important at pH 2 than at pH 7. The role of helix propensity in stabilizing the different forms of the molten globule state is examined and it is shown that it cannot account for the differences. The strikingly different behavior observed at pH 2 and 7 indicates that the A-state may not be a rigorous mimic of the folding intermediate populated at pH 7.     

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection‐site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5–100 μM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross‐linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 μM insulin. These findings suggest that oligomers may be on‐ and off‐pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer‐sized aggregates, which may be of physiological relevance and provides insight into injection‐site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.     

Small‐angle X‐ray scattering of BAMLET at pH 12: A complex of α‐lactalbumin and oleic acid

BAMLET (Bovine Alpha‐lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET‐like complexes, a novel class of protein‐based anti‐cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X‐ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo‐α‐lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo‐α‐lactalbumin was performed and the results suggest that α‐lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non‐specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure. Proteins 2014; 82:1400–1408. © 2014 Wiley Periodicals, Inc.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Stability of proteins in the presence of polyols estimated from their guanidinium chloride-induced transition curves at different pH values and 25 degrees C

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.     

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

AIMS: Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. METHODS AND RESULTS: Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. CONCLUSIONS: A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.