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.     

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.     

Trifluoroethanol-induced beta --> alpha transition in beta-lactoglobulin: hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion

Alcohols, such as 2,2,2-trifluoroethanol (TFE), have been shown to induce a cooperative transition to an open helical structure in many proteins, but the underlying molecular mechanism has not been identified. Here, we employ the technique of magnetic relaxation dispersion (MRD) to study the TFE-induced beta --> alpha transition of beta-lactoglobulin at pH 2.4. Unlike traditional techniques that focus on protein secondary structure, the MRD method directly monitors the solvent, providing quantitative information about preferential solvation and solvent penetration and about the overall size and structural integrity of the protein. In this multinuclear MRD study, we use the (2)H and (17)O resonances to examine hydration and the (19)F resonance to study TFE. The transformation from the native to the helical state via an intermediate state at 300 K is found to be accompanied by a progressive expansion of the protein and loss of specific long-lived hydration sites. The observation of (17)O and (19)F dispersions from the helical state shows that water and TFE penetrate the protein. The MRD data indicate a strong accumulation of TFE at the surface as well as in the interior of the protein. At 277 K, BLG is much less affected by TFE, remaining in the native state at 16% TFE, but adopting a nonnative structure at 30% TFE. This nonnative structure is not penetrated by long-lived water molecules. The implications of these findings for the mechanism of TFE-induced structural transformations are discussed.     

2,2,2-Trifluoroethanol-Induced structural change of peanut agglutinin at different pH: A comparative account

Peanut Agglutinin (PNA) is a legume lectin with a unique open quarternary structure. It is a homotetrameric protein, the monomeric subunit of which is made up of 3 beta sheets. The structural change in this protein has been induced by 2,2,2-trifluoroethanol (TFE) at two different pH. At neutral pH, PNA exists as a homotetramer, while at pH 2.5, it is known to dissociate to a dimer. The effect of TFE has been studied at both the pH by intrinsic tryptophan fluorescence, far and near UV Circular Dichroism, ANS binding and dynamic light scattering. At low pH, 15% TFE is found to induce a molten globule like state that shows maximum ANS binding. Increasing concentration of TFE increases alpha helical content and the compactness of the protein. The compact PNA at higher concentration of TFE is structurally different from the native structure. The effect of TFE at neutral pH on PNA is somewhat different from that observed at low pH. TFE does not induce molten globule like state at this pH. The detailed study of the structural change of PNA by TFE has been presented.     

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.     

Protein precipitation and denaturation by dimethyl sulfoxide

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.     

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.     

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.     

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.     

Conformational polymorphism, stability and aggregation in spider dragline silks proteins

Spider silk is spun in a complex and unique process, thought to depend on a hydrophobic conversion of a predominantly disordered to a beta-sheet rich protein structures. To test this hypothesis we monitored the effect of cationic (DOTAC) and anionic (alkyl sulfate) detergents and of (ii) solvent polarity using a series of alcohols on the secondary structure transition in dilute solutions of native spidroin. Our results showed that the detergents hydrophilic head charge and hydrophobic tail length cooperatively induced either a transition to the beta-sheet rich form or a stable helical state. Changing the solvent polarity showed that HFIP and TFE induced formation of stable helical forms whereas MeOH, EtOH and IsoP induced a kinetically driven formation of beta-sheet rich structure.     

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.     

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative studies on trifluoroethanol (TFE) state of a thermophilic alpha-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial alpha-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the alpha-amylases, a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the alpha-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease.     

Organic solvent mediated self-association of an amyloid forming peptide from beta2-microglobulin: an atomic force microscopy study

Human beta(2)-microglobulin (beta(2)m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the beta strands of beta(2)m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (Pbeta(2)m) spanning one of the beta-strands of human beta(2)-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that Pbeta(2)m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, beta-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of Pbeta(2)m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

Structural intermediates of acid unfolded Con-A in different co-solvents: fluoroalcohols and polyethylene glycols

A molten globule-like intermediate of Con-A was obtained when subjected to acid unfolding. At pH 2 the intermediate was found to have native-like secondary structure, somewhat denatured tertiary structure and maximum ANS binding. Further the stability of this intermediate was studied in presence of fluoroalcohols (TFE and HFIP) and polyethylene glycols (PEG-400, 4000 and 20,000). Secondary structural changes were monitored by far-UV CD while alterations in the tertiary structure of the acid unfolded intermediate were probed by near-UV CD. To study the environment and position of the tryptophan residues present intrinsic fluorescence studies were performed. ANS binding studies were also made to know the extent of exposure of the hydrophobic patches. Using the above-mentioned techniques it was found that in presence of fluoroalcohols the pH 2 intermediate was transformed to a state with predominant alpha-helical secondary and denatured tertiary structures. In the pathway of these transformations MG-like intermediates were formed at 10% TFE and 6% HFIP. The folding intermediate of Con-A obtained at pH 2 underwent a series of conformational changes when exposed to different molecular weight PEGs. Secondary structure was induced by low molecular weight PEG-400 and low concentrations of PEG-4000 and PEG-20,000 while at higher concentrations transition in structure was observed. Tertiary structure was stabilized only at low concentrations of PEG-400. PEG-4000 and PEG-20,000 in the whole concentration range resulted in the loss of tertiary structure.     

Deceleration of Arginine Kinase Refolding by Induced Helical Structures

Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for −2.19 kcal/mol for AutoDock4.2 and −20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.     

Fluorinated alcohol, the third group of cosolvents that stabilize the molten-globule state relative to a highly denatured state of cytochrome c

The effects of 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP) on the conformation of cytochrome c (cyt c) at pH 1.9 were studied using a combination of spectroscopic and physical methods. Analysis varying the HFIP concentration showed that a compact denatured conformation (M(HF)) accumulates in a low concentration range of HFIP in the middle of structural transition from the highly unstructured acid-denatured state to the highly helical alcohol-denatured state of cyt c. This contrasts clearly with the effect of isopropanol (IP), in which no compact conformation accompanied with the transition. Analysis varying concentrations of HFIP and NaCl concurrently showed that the M(HF) state of cyt c is essentially identical to the salt-induced molten-globule (M(G)) state, and the M(G) state in the presence of salt was also stabilized by a low concentration of HFIP. Furthermore, 2,2,2-trifluoroethanol stabilized M(HF) similarly to HFIP, supporting the proposition that the specific effect observed for HFIP is caused by fluorination of alcohol. The mechanism stabilizing compact conformation by HFIP remains unclear, but is probably distinct from that of salts and polyols, which are also known to stabilize the M(G)-like state.     

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.