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.     

Characterization of a common intermediate of pea lectin in the folding pathway induced by TFE and HFIP

When pea lectin was exposed to a low pH range, it was found that the secondary structure of the lectin resisted conformational changes to a large extent up to pH 2.4 and below this pH, a sharp transition was observed which could be due to the presence of 27 acidic amino acid residues present in the protein. The effects of 1,1,1,3,3,3 hexafluoro-isopropanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) on the conformation of pea lectin at pH 2.4 were studied using circular dichroism and fluorescence spectroscopy. Analysis varying the TFE concentration showed that up to 80% TFE (v/v) protein retained the residual beta-structure accompanied by a loss in tertiary structure. A similar conformation is presumed to exist at 4% HFIP (v/v), with an increase in HFIP concentration structural rearrangements occurred and a transition from beta-structure to alpha-helical structure started from 12% HFIP which completed at 30% HFIP. Our studies show the occurrence of a common intermediate in the folding pathway of pea lectin induced by two different fluoroalcohols, which differ in their mode of action to stabilize the secondary structure of a given protein. While TFE was not found to induce any alpha-helical structure, HFIP caused the transition of pea lectin, which is predominantly a beta-sheet protein, to a structure rich in alpha-helical contacts. Thus, our results also point out the possibility of a non-hierarchical model of protein folding in lectins.     

Dissolution of beta2-microglobulin amyloid fibrils by dimethylsulfoxide

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on beta(2)-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.     

Conformational properties of methionine homo-oligopeptides in solution

A conformational analysis was carried out in solution on a series of L -methionine oligomers having the general formula \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm BOC\rlap{--} (L - Met\rlap{--})}_n {\rm OMe (}n = 2 - 7)$\end{document}. We examined these oligopeptides in TFE, HFIP, EG, and mixed organic–water media. The critical size for helix formation was found to be seven residues in TFE, whereas the β-associated structure appears at the pentamer in EG and TFE–water (20 : 80, v/v). In HFIP, however, the oligomers exist essentially in an unordered conformation.     

Interactions of trifluroethanol with the Trp-cage peptide

It has been suggested that aggregation of fluorinated alcohols in water solutions is involved with the abilities of these alcohols to provoke conformational changes in peptides and proteins. The extent of fluoroalcohol aggregation depends on the degree of fluorination: hexafluoroisopropanol (HFIP) is more extensively aggregated than is TFE. We previously described a study of the interactions of HFIP with the peptide Trp-cage and provided evidence for the formation of long-lived complexes between this fluoroalcohol and the peptide. In the present work, we have examined the interactions of the less-fluorinated TFE with Trp-cage, in order to probe the role of fluoroalcohol aggregation in the phenomena observed. Intermolecular (1)H{(19)F} nuclear Overhauser effects arising from interactions of TFE with the hydrogens of the peptide in a solution containing 42% TFE were determined at sample temperatures from 5 to 45 degrees C. It is shown that the folded state of the peptide under these conditions is essentially the same as that observed in water and in 30% HFIP-water. The observed peptide-solvent NOEs indicate formation of complexes of Trp-cage with TFE that persist for times of the order of 1 ns. The interactions leading to complexes with TFE are somewhat weaker than those involved in complex formation with HFIP. There are no indications that the aggregation of fluoroalcohol is a necessary concomitant of the interactions of TFE or HFIP with Trp-cage. Rather, the stronger and more long-lived interactions of HFIP with Trp-cage appear to be primarily the result of the greater hydrogen-bonding ability and hydrophobicity of this fluoroalcohol.     

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.     

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.     

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-Trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more α-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivation indicated that the active site of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol.

Alcohols denature the native state of proteins, and also stabilize the alpha-helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine beta-lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, beta-lactoglobulin exhibited a transformation from the native state, consisting of beta-sheets, to the alpha-helical state, whereas melittin folded from the unfolded state to the alpha-helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol-induced transitions were analyzed assuming a two-state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle-like clusters of HFIP is important.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Induction of 'molten globule' like state in acid-denatured state of unmodified preparation of stem bromelain: implications of disulfides in protein folding

A denatured state of unmodified preparation of stem bromelain representing a structureless form has been characterized at pH 2.0 and the effect of increasing concentration of TFE on the acid-denatured state has been investigated by circular dichroism (CD), fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show considerable accumulation of secondary structure when the acid-denatured bromelain is subjected to 70% (v/v) TFE and exhibited close resemblance to spectral features of those of pH 7.0 preparation. Interestingly, the acid-denatured state also regained some tertiary structure/interactions, with increasing concentration of TFE and at 60% (v/v) TFE, these approached almost those of the native like state. However, further increase to 70% (v/v) TFE resulted in complete loss of tertiary structure/interactions. Tryptophan fluorescence emission studies also suggested the induction of significant compact structure at 60% (v/v) concentration of TFE. In addition the acid-denatured state showed enhanced binding of ANS in presence of 60% (v/v) TFE. Taken together these observations suggest the existence of a molten globule state in acid-denatured bromelain between 60 and 70% (v/v) TFE. A similar molten globule state under identical conditions has been identified in reduced and carboxymethylated preparation of stem bromelain as reported in our earlier communication [Arch. Biochem. Biophys. 413 (2003) 199]. Comparison suggests unfolding/folding behavior of the bromelain to be independent of the intactness of the disulfide bonds.     

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations and a retention of a native-like structure - but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of . Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in . The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.     

Cosolvents Induced Unfolding and Aggregation of Keyhole Limpet Hemocyanin

The objective of this study was to examine the effects of 2,2,2 trifluoroethanol (TFE) and acetonitrile (ACN) on the stability, behavior, and structural characteristics of giant multimeric protein Keyhole Limpet hemocyanin (KLH) by combining the circular dichroism (CD) and fluorescence measurements of KLH solution. In concentration range 20–50 % (v/v) TFE, protein at pH 7.4 shows visible aggregation while no aggregation was observed in the entire concentration range of TFE at molten globule (MG) state (pH 2.8) and resulted in stable α-helix. Our result shows that in the presence of 80 % (v/v) and 40 % (v/v) TFE, at native (pH 7.4) and MG state (pH 2.8) occurred in a highly helical state referred to as TFE denatured state I and II, respectively. However, in case of ACN, aggregation starts above 40 % (v/v) for pH 7.4 and at 80 % (v/v) for acid-induced MG (pH 2.8) state, which was dominated by β-sheet structure and referred to as ACN denatured state III and IV. An important object of our investigation is to get more detail study of efficiency of cosolvents in inducing structural changes in KLH. The dependence of scattering intensity and the R _h on alcohol concentrations was investigated at 25 °C.     

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions

The conformational transitions of bovine beta-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of beta-lactoglobulin A from the native to the highly helical state is clearly demonstrated.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

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.