Cooperative α‐helix formation of β‐lactoglobulin and melittin induced by hexafluoroisopropanol

Abstract

Alcohols denature the native state of proteins, and also stabilize the a-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 P-lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, P-lactoglobulin exhibited a transformation from the native state, consisting of P-sheets, to the a-helical state, whereas melittin folded from the unfolded state to the a-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 rn value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the rn 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.

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

“…Another force that expands the protein conformation also acts, however, in the alcohol solution mainly because of the nonpolar character of alcohol~Nozaki & Tanford, 1971;Thomas & Dill, 1993;Liu & Bolen, 1995!. A compromise between the two conflicting forces is a probable reason why the highly helical but expanded conformation has been observed in alcohol solutions~Kamatari et al, 1996;Hoshino et al, 1997!. In a low concentration range of HFIP, however, the force minimizing exposure of main-chain components is probably more dominant than the force that expands the denatured chain, which could explain the stabilization of the compact denatured state of cyt c under this condition. The view that HFIP at a low concentration exerts an exceptionally strong intrachain attraction is consistent with many previous observations including stronger helical induction of HFIP than alkyl alcohols~Arvinte & Drake, 1993; Hirota et al, 1997!, assembly of short peptides~Mchaourab et al, 1993Cort et al, 1994! and specific stabilization of a folding intermediate of acylphosphatase at low HFIP concentrations~Chiti et al, 1999!.…”

“…Thermodynamic force inducing secondary structure of protein may also collapse a denatured protein chain by enhancing intrachain hydrogen bonding, and thus some special alcohols having exceptionally strong helical induction may stabilize a compact state by overcoming the expanding force exerted by the nonpolar character of alcohol. A candidate alcohol of this type is 1,1,1,3,3,3-hexafluoro-isopropanol ~HFIP!, which strongly induces a-helices and aggregation of proteins and peptides~Mchaourab et al, 1993;Cort et al, 1994;Cort & Andersen, 1997;Hirota et al, 1997!. A question that arises is whether a compact conformation is stabilized when HFIP is added to the solution of a highly unstructured denatured state of a protein, cytochrome c~cyt c!.…”

Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk

“…First, fluorinated alcohols are capable of inducing helical structuring 62,63 . As such, the observed secondary structuring could be argued to be favored by the solvent environment.…”

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: A calorimetric and spectroscopic study

“…Investigations of such conformational changes have provided valuable information about the role of the solvent in the maintaining the native, intermediate, or denatured state of a given protein. Recently, numerous papers studying the influence of different solvents especially alcohols, on the formation of a-helical intermediates have been published with the common goal of better understanding the protein folding pathways~Bych-kova et al, 1996;Jayaraman et al, 1996;Kamatari et al, 1996;Hirota et al, 1997!. It has been shown by CD spectroscopy at isothermal conditions that alkylureas similarly to alcohols~Hi-rota et al, 1997! induce a formation of a-helical secondary structure in b-lactoglobulin A~b-lg!~Lapanje & Kranjc, 1982!…”