Boolean Logic Gate Operation in Bacteriorhodopsin of Purple Membrane Based on a Molten Globule-like State.

Bacteriorhodopsin (bR) of purple membrane (PM) has increasing technical interests, particularly in photonic devices and bioelectronics. The present work has concerned with monitoring the temperature dependence of passive electric responses in-plane and out-of-plane of the membranes. Based on thermal properties observed orthogonally here for PM, a high-temperature intermediate of bR has been suggested to populate at around 60 °C, which may be ascribed to a molten globule-like state. This intermediate has been found to be enclosed between two reversible thermal transitions for PM. Large-scale turnover in the energy of activation, for these two thermal transitions, occurs steeply at such state at 60 °C, above which does bR reverse the sign of dielectric anisotropy (i.e. crossover) provided the operating frequency should be above the crossover frequency, at which the reversal occurs. No such crossover was found to occur below the crossover frequency, even above the crossover temperature (i.e. 60 °C). Likewise, no such crossover was found to occur below the crossover temperature, even above the crossover frequency. Relying on this reasoning, a logic gate operation may be declared implicating bR for bioelectronics and sense technological relevance. In addition, the results specify "dual frequency" as well as "dual temperature" characteristics to bacteriorhodopsin.

Carboxymethyl chitosan coating infused with linalool-loaded molten globular ß-Lactoglobulin nanoparticles for extended preservation of fresh-cut apples.

This investigation employed molten globule state ß-lactoglobulin nanoparticles (MG-BLGNPs) for encapsulating linalool (LN) combined with carboxymethyl chitosan (CMC) coating to enhance the shelf-life of fresh-cut apples. The effect of different MG structures on the encapsulation efficiency of BLGNPs and the properties of coating was studied. Structural characterization and molecular simulation showed structural differences between heat-induced MG state (70-BLGNPs, heated at 70 °C for 1 h) and sodium dodecyl sulfate-co-heat-induced MG state (SDS/70-BLGNPs, treated with 0.192 mg/mL SDS for 10 min, then heated at 70 °C for 1 h), with the latter being more unfolded. LN self-assembles into MG-BLGNPs, among the generated particles, SDS/70-BLG@LN exhibits stronger binding effect and higher LN loading capacity. Integration of MG-BLG@LN into CMC enhanced coating's mechanical properties and adhesion to fresh-cut apples. The SDS/70-BLG@LN/CMC coating showed superior preservation on fresh-cut apples during storage, reducing enzymatic browning, membrane lipid oxidation, and microbial growth while maintaining hardness and overall quality.

Influence of divalent metal cations on α-lactalbumin fibril formation.

The effect of binding of divalent metal cations (Ca2+, Cu2+, Mg2+, Mn2+, Zn2+) on the kinetics of fibril formation of bovine α-lactalbumin at acidic conditions is considered. The kinetic parameters of the process were determined using a thioflavin T fluorescence assay. The DSC thermograms of bovine α-lactalbumin in the presence and absence of cations were recorded. The duration of the lag period correlates with the changes in the thermal stability of the molten globule of the protein in the presence of cations. The final thioflavin T fluorescence intensity after formation of the mature fibrils decreases under the influence of calcium ions which strongly bind to the monomeric protein, and increases in solutions containing copper and especially zinc. These ions seem to accelerate secondary nucleation processes and change the fibril morphology, which was confirmed by atomic force microscopy imaging.

Slow Misfolding of a Molten Globule form of a Mutant Prion Protein Variant into a ß-rich Dimer.

Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a ß-rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a ß-rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the ß-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Inside of the burst-phase intermediate of a protein folding. Hydration of hydrophobic groups.

The thermal effect of the formation of the "burst-phase" folding intermediate has been studied using a titration calorimeter. It is shown that, unlike the total thermal effect of native structure formation, it can be both positive and negative depending on the temperature. The reasons for this paradoxical behavior are analyzed. A conclusion is drawn about the leading role of dehydration of non-polar groups in the first stage of folding.

Proline Peptide Bond Isomerization in Ubiquitin Under Folding and Denaturing Conditions by Pressure-Jump NMR.

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis â†’ trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.

Conformational stability of peroxidase from the latex of Artocarpus lakoocha: influence of pH, chaotropes, and temperature.

The latex of the medicinal plant Artocarpus lakoocha (A. lakoocha), which has been shown to have potential anti-inflammatory and wound-healing capabilities, contains a novel heme-peroxidase. This protein was subjected to activity assays, fluorescence spectroscopy, and far-UV circular dichroism to investigate its structure, dynamics, and stability. The results demonstrated the presence of three folding states: the native state (N) at neutral pH, intermediate states including molten globule (MG) at pH 2 and acid-unfolded (UA) at pH 1.5 or lower, and acid-refolded (A) at pH 0.5, along with alkaline denatured (UB) at pH 8-12 and the third denatured state (D) at GuHCl concentrations exceeding 5 M. Absorbance studies indicated the presence of loosely associated form of heme in the pH range of 1-2. The protein showed stability and structural integrity across a wide pH range (3-10), temperature (70°C), and high concentrations of GuHCl (5 M) and urea (8 M). This study is the first to report multiple 'partially folded intermediate states' of A. lakoocha peroxidase, with varying amounts of secondary structure, stability, and compactness. These results demonstrate the high stability of A. lakoocha peroxidase and its potential for biotechnological and industrial applications, making it a valuable model system for further studies on its structure-function relationship.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

Mechanistic Insight into Poly-Reactivity of Immune Antibodies upon Acid Denaturation or Arginine Mutation in Antigen-Binding Regions.

The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can occur in individual molecules of natural serum antibodies, likely due to their conformation flexibility, and, for therapeutic antibodies, it plays a critical role in their clinical development. On the one hand, it can enhance their binding to target antigens and cognate receptors, but, on the other hand, it may lead to a loss of antibody function by binding to off-target proteins. Notably, poly-reactivity has been observed in antibodies subjected to treatments with dissociating, destabilizing or denaturing agents, in particular acidic pH, a common step in the therapeutic antibody production process involving the elution of Protein-A bound antibodies and viral clearance using low pH buffers. Additionally, poly-reactivity can emerge during the affinity maturation in the immune system, such as the germinal center. This review delves into the underlying potential causes of poly-reactivity, highlighting the importance of conformational flexibility, which can be further augmented by the acid denaturation of antibodies and the introduction of arginine mutations into the complementary regions of antibody-variable domains. The focus is placed on a particular antibody's acid conformation, meticulously characterized through circular dichroism, differential scanning calorimetry, and sedimentation velocity analyses. By gaining a deeper understanding of these mechanisms, we aim to shed light on the complexities of antibody poly-reactivity and its implications for therapeutic applications.

The Origin of Discrepancies between Predictions and Annotations in Intrinsically Disordered Proteins.

Disorder prediction methods that can discriminate between ordered and disordered regions have contributed fundamentally to our understanding of the properties and prevalence of intrinsically disordered proteins (IDPs) in proteomes as well as their functional roles. However, a recent large-scale assessment of the performance of these methods indicated that there is still room for further improvements, necessitating novel approaches to understand the strengths and weaknesses of individual methods. In this study, we compared two methods, IUPred and disorder prediction, based on the pLDDT scores derived from AlphaFold2 (AF2) models. We evaluated these methods using a dataset from the DisProt database, consisting of experimentally characterized disordered regions and subsets associated with diverse experimental methods and functions. IUPred and AF2 provided consistent predictions in 79% of cases for long disordered regions; however, for 15% of these cases, they both suggested order in disagreement with annotations. These discrepancies arose primarily due to weak experimental support, the presence of intermediate states, or context-dependent behavior, such as binding-induced transitions. Furthermore, AF2 tended to predict helical regions with high pLDDT scores within disordered segments, while IUPred had limitations in identifying linker regions. These results provide valuable insights into the inherent limitations and potential biases of disorder prediction methods.

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 µs Molecular Dynamics Simulation.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 µs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

Features of Protein Unfolding Transitions and Their Relation to Domain Topology Probed by Single-Molecule FRET.

A protein fold is defined as a structural arrangement of a secondary structure in a three-dimensional space. It would be interesting to know whether a particular fold can be assigned to certain features of the corresponding folding/unfolding transitions. To understand the underlying principles of the manifold folding transitions in more detail, single-molecule FRET is the method of choice. Taking the two-domain protein phosphoglycerate kinase (PGK) as an example, we investigated denaturant-induced unfolded states of PGK using the above method. For this purpose, different intramolecular distances within the two domains were measured. In addition to the known two-state transition, a transition with a compact folding intermediate was also identified in each of the two domains. Based on the structural homology of the domains (characterized by a Rossmann fold) and the striking similarity in the features of the measured distance changes during unfolding, clear evidence emerged that the underlying domain topology plays an important role in determining the observed structural changes.

The role of hydrophobic interactions in the molten globule state of globular protein modulated by surfactants.

In order to highlight the role of hydrophobic interactions in the molten globule (MG) state of globular protein modulated by surfactants, the interactions of bovine α-lactalbumin (α-LA) with alkyl trimethylammonium bromides (CnTAB, n = 10, 12, 14, and 16) have been studied by experimental and theoretical techniques. Isothermal titration calorimetry (ITC) showed that the enthalpy changes (ΔH) and area of the enthalpogram increased with increasing the chain length of CnTAB. The result of fluorescence, circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectrum suggested that C10TAB and C12TAB unfolded α-LA partially, C14TAB reconstructed protein with a native-like secondary structure content, and C16TAB induced an MG state α-LA. The SAXS results confirmed that the tertiary structure of α-LA was disrupted by C16TAB forming an MG state complex with a micelle-like structure even at the surfactants concentrations below CMC. As indicated by MD results, the ß-domain and unstructured region(s) were involved in the MG state α-LA modulated by CnTAB. This work not only provides molecular insights into the role of hydrophobic interactions in the MG state of a globular protein but also helps understand the mechanism of preparing α-LA based biomacromolecule modulated by hydrophobic interactions.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

Do "Newly Born" orphan proteins resemble "Never Born" proteins? A study using three deep learning algorithms.

"Newly Born" proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by the expression of novel open reading frames, and appear throughout evolution. We were curious if three recently developed programs for predicting protein structures, namely, AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such "Newly Born" proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called "Never Born" proteins. The programs were used to compare the structures of two sets of "Never Born" proteins that had been expressed-Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, although the models generated were scored as being of low quality, they nevertheless revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms, in agreement with the experimental data, whereas the members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins, again consistent with the experimental data. These predicted differences were shown to be statistically significant by comparing their accessible surface areas. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Surprisingly, only for the protein which did not have a novel fold, and was taxonomically restricted, rather than being a true orphan, did all three algorithms predict very similar, high-quality structures, closely resembling the crystal structure. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with only two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member, and to the fact that the Dali server revealed several nonrelated proteins with similar folds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Proteins:3.

Hen lysozyme fibrillogenesis, molten globule intermediate and effect of copper salts.

The amyloid fibres have been related to many diseases. The molten globule intermediate has been proposed to form part of the folding pathway of many proteins. In the present study, we investigated the mechanism of amyloid-fibres formation of hen egg-white lysozyme (HEWL) incubated in a potassium phosphate buffer, pH 11.8, 100 mM, at 37 °C for 30 h, and evaluated the influence of Cu(II) present in two salts (CuSO4 and CuCl2) during fibrillogenesis. Co-incubation and post-incubation of lysozyme with copper salts reduced the fluorescence signal of thioflavin T with an increment in the intrinsic fluorescence of the protein. The ANS fluorescence test showed that incubation of HEWL for 6 h generated a molten globule intermediate state that formed amyloid fibres when incubation was carried out for a 30-h timespan. Dynamic light scattering showed a heterogeneous population of states in samples incubated in the absence or the presence of salts during the fibrillation process. The existence of a reducing potential was verified during the formation of HEWL amyloid fibres with the bathocuproine disulphonate test. Transmission electron microscopy confirmed the presence and absence of fibres in solutions incubated with and without Cu(II). This work demonstrated that lysozyme formed amyloid fibres at 37 °C and copper inhibited its formation.Communicated by Ramaswamy H. Sarma.

The interaction of trehalose and molten globule state soybean 11S globulin and its impact on foaming capacities.

BACKGROUND: Soybean 11S globulin has good functional properties, which are widely used in the field of food. However, natural soybean 11S globulin (N-11S) has low flexibility and is easy to aggregate, impacting its foaming process. Studies have shown that soybean 11S globulin in molten globule state (MG-11S) has better molecular flexibility than N-11S, and trehalose has been shown to improve the properties of proteins. Therefore, this study investigated the interaction mechanism between trehalose and MG-11S, and its impact on rheological and foaming properties of MG-11S. RESULTS: The molecular docking and intrinsic fluorescence results showed that hydrogen bonding was the main interaction force at lower than 0.5 mol L-1 trehalose added. Meanwhile, rheology and foaming showed that the MG-11S-trehalose complexes had better viscoelasticity, foaming ability (66.67-86.67%) and foaming stability (75.00-89.29%) than N-11S (16.67% foaming ability and 40.00% foaming stability); however, when the trehalose was higher than 0.5 mol L-1 , molecular crowding occurred and H-bonds were weakened, resulting in reduction of foaming capacities. Microstructure determination showed that trehalose attached to the surface of foam membrane; meanwhile, the foaming structure of the complex with 0.5 mol L-1 trehalose had a thicker liquid film with decreased drainage rate, less agglomeration and disproportionation of foam, illustrating the best foaming ability and foaming stability. CONCLUSION: The results suggested that trehalose at different concentrations can interact with MG-11S through different mechanisms, and improve the foaming capacity of MS-11S. This provided a reference for the application of MS-11S in foaming food. © 2022 Society of Chemical Industry.

Structural Transitions of Alpha-Amylase Treated with Pulsed Electric Fields: Effect of Coexisting Carrageenan.

Pulsed electric field (PEF) is an effective way to modulate the structure and activity of enzymes; however, the dynamic changes in enzyme structure during this process, especially the intermediate state, remain unclear. In this study, the molten globule (MG) state of α-amylase under PEF processing was investigated using intrinsic fluorescence, surface hydrophobicity, circular dichroism, etc. Meanwhile, the influence of coexisting carrageenan on the structural transition of α-amylase during PEF processing was evaluated. When the electric field strength was 20 kV/cm, α-amylase showed the unique characteristics of an MG state, which retained the secondary structure, changed the tertiary structure, and increased surface hydrophobicity (from 240 to 640). The addition of carrageenan effectively protected the enzyme activity of α-amylase during PEF treatment. When the mixed ratio of α-amylase to carrageenan was 10:1, they formed electrostatic complexes with a size of ~20 nm, and carrageenan inhibited the increase in surface hydrophobicity (<600) and aggregation (<40 nm) of α-amylase after five cycles of PEF treatment. This work clarifies the influence of co-existing polysaccharides on the intermediate state of proteins during PEF treatment and provides a strategy to modulate protein structure by adding polysaccharides during food processing.

Polyol and sugar osmolytes stabilize the molten globule state of α-lactalbumin and inhibit amyloid fibril formation.

Protein misfolding and aggregation are associated with several human diseases such as Alzheimer's, Parkinson's, prion related disorders, type-II diabetes, etc. Different strategies using molecular chaperones, synthetic and naturally occurring small molecules, osmolytes, etc. have been used to prevent protein aggregation and amyloid fibril formation. In this study, we have used bovine α-lactalbumin at pH 1.6, 37 °C, and shaking conditions to promote amyloid fibril formation. Polyol and sugar osmolytes like glycerol, sorbitol, and trehalose have been used to inhibit the fibrillation of a number of proteins. In the present work, amyloid fibril formation of α-lactalbumin has been shown by ThT assay and AFM, while changes in the secondary structure during fibrillation has been followed by circular dichroism spectroscopy. Our results show that glycerol, sorbitol, and trehalose affect amyloid fibril formation of α-lactalbumin in a concentration-dependent manner. There is a delay in the lag phase of amyloid fibril formation in sorbitol and trehalose and complete inhibition in 6 M glycerol. Our results indicate that delay in the lag phase and inhibition of amyloid fibril formation are due to the stabilization of molten globule state by these osmolytes. At pH 1.6, the molten globule as well as the amyloid fibrils bind to ANS. However, when pH was shifted from 1.6 to 7, only the oligomeric and the fibrillar species bind to ANS due to refolding of molten globule state. The outcome of this study might be useful in designing small molecules which may stabilize the intermediate states, thus preventing amyloid fibril formation.