Trifluoroethanol‐induced conformational transitions of proteins: Insights gained from the differences between α‐lactalbumin and ribonuclease A

Abstract

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.

Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

“…In contrast, the molar ellipticity of the R9AP-Cter peptide is much smaller in HFIP than in HMeOH (Fig. 2G), which is not consistent with previous observations that this solvent favors the formation of α-helical structures (see section 3.1) [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103].…”

“…Nevertheless, care must be taken when using TFE and HFIP because it is generally believed that they enhance the population of helical conformations of peptides and proteins. In particular, since the pioneering work of Tamburro et al [88], fluorinated alcohols, such as TFE and HFIP, have been shown to promote the formation of α-helices for a large number of proteins and peptides (see, for example, references [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] and the references cited therein). It has been proposed that fluoroalcohol complexes would displace water molecules from the surface of the peptide; the acidity or hydrogen-bonding ability and hydrophobicity of the fluoroalcohols appear to play an important role [104].…”

“…2F does not fit well with any of the infrared spectra of this peptide; indeed, the spectrum in HFIP indicates a long and rigid α-helix whereas that in H-MeOH and the powder respectively reveal a large content in turns and β-sheets. This raises also the issue that HFIP could favor the formation of α-helical structures in the case of this peptide [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103].…”

A Circulating β₂-Microglobulin Intermediate in Hemodialysis Patients

“…β 2 m has a hydrophobic core and is rich in intramolecular hydrogen bonds [19, 20], which favors modification by organic solvents, such as acetonitrile or trifluoroethanol [18, 21]. A detailed study by Heegaard et al [12] and Chiti et al[ 18] proved that I 2 -β 2 m was more amyloidogenic than the N-β 2 m, and treatment with acetonitrile induces a reversible conformational variant of β 2 m, I 2 -β 2 m. This study showed a comparable result.…”

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

“…Alcohol destabilizes hydrophobic cores of protein because of its nonpolar character while it enhances secondary-structure formation of protein by minimizing exposure of peptide backbone~Nozaki & Tanford, 1971;Thomas & Dill, 1993;Liu & Bolen, 1995!. These alcohol effects lead to destabilization of the native state of protein and induction of a highly helical denatured conformation~Fink & Painter, 1987;Dyson et al, 1992;Buck et al, 1993;Alexandrescu et al, 1994;Kippen et al, 1994;Shiraki et al, 1995;Kamatari et al, 1996;Hoshino et al, 1997;Gast et al, 1999!. Studies on alcohol effects provide insights into biologically important events because the alcohol solution mimics the environment of biomembrane~Bychkova et al, 1996!, modifies folding pathways of proteins~Lu et al, 1997;Chiti et al, 1999!…”