Ultrasonic Studies of Alcohol-Induced Transconformation in β-Lactoglobulin: The Intermediate State

Abstract

In mixed alcohol-water solvents, bovine beta-lactoglobulin undergoes a cooperative transition from beta-sheet to a high alpha-helix content conformer. We report here the characterization of beta-lactoglobulin by compressibility and spectroscopy measurements during this transconformation. Both the volume and compressibility increase as a function of alcohol concentration, up to maximal values which depend on the chemical nature of the three alcohols used: hexafluoroisopropanol, trifluoroethanol, and isopropanol. The order of effectiveness of alcohols in inducing the compressibility transition is identical to that previously reported for circular dichroism and thus independent of the observation technique. The highly cooperative sigmoidal curves found by compressibility determination match closely those obtained by circular dichroism at 222 nm, indicating a correlation between the two phenomena measured by the two different techniques. The presence of an equilibrium intermediate form was shown by the interaction of beta-lactoglobulin with 8-anilino-1-naphthalene sulfonic acid, a probe widely used to detect molten-globule states of proteins. It was correlated with the plateau region of the volume curves and with the inflexion points of the sigmoidal compressibility curves. Ultrasound characterization of proteins can be carried out in optically transparent or nontransparent media.

2,2,2-Trifluoroethanol Changes the Transition Kinetics and Subunit Interactions in the Small Bacterial Mechanosensitive Channel MscS

“…The ability for TFE to stabilize helical conformations in peptides and accelerate protein folding has been explained by aggregation of TFE around the protein backbone, local exclusion of water from the competition for hydrogen bonds, and possibly by lowering the effective dielectric constant of the solvent (36). This mechanism is consistent with TFE's tendency to form microscopic clusters in aqueous solutions (1,9), partition into hydrophobic protein crevices (34), and promote desolvation of protein surfaces that normally form buried contacts (14,36). At the same concentrations (15-30 vol %) that stabilize soluble proteins, TFE completely disrupts KcsA and MscS as well as many other membrane complexes (17,21).…”

“…1 C, lanes 6-11). Apparently, even low amounts of TFE are sensed by the protein, causing it to migrate more slowly, likely due to the effect of ''swelling'' of hydrophobic cavities and voids (34).…”

Study of the binding between lysozyme and C10-TAB: Determination and interpretation of the partial properties of protein and surfactant at infinite dilution

“…The volumetric thermodynamic properties (volume and its derivatives with respect to temperature, expansibility, and with respect to pressure, compressibility) have been widely employed in the study of folding/unfolding transitions due to changes in temperature [4][5][6][7], pressure [6,8], pH [7,[9][10][11][12][13], cosolvent composition [14,15], oxidation/ reduction reactions [16] and binding of ligand [17][18][19][20][21][22][23] because these properties are sensitive to the solute-solvent interaction (hydration) and to the intrinsic packing. In this respect, it has been suggested that the "efficacy of the use of volumetric measurements for solving problems of biological relevance ultimately depends on our ability to rationalize measured volumetric observables in terms of various volumetric inter-and intramolecular interactions including, but not limited to, hydration and intrinsic packing" [24].…”

Secondary‐structure analysis of alcohol‐denatured proteins by vacuum‐ultraviolet circular dichroism spectroscopy

“…Alcohols disrupt the tertiary structure of a protein by exposing the internal hydrophobic residues to the solvent, but the denatured protein is rich in α‐helices, in contrast to other types of denaturation mainly disrupting α‐helices and producing considerable amounts of β‐strands, turns, and poly‐ L ‐proline type II helices (PPIIs) 11–14. One conclusion of the recent studies is that the alcohol‐denatured protein forms an open helical structure with strong local interactions within each helix but weak interactions between helical segments,15 unlike the compact molten globule state stabilized by the interhelix hydrophobic interactions 16, 17. However, although there is some evidence for the existence of β‐strands,9, 18, 19 the general features of the secondary structures (i.e., their contents, numbers of segments, and sequences) of alcohol‐denatured proteins remain unclear because of the technical difficulties of studying highly fluctuating structures.…”

“…The structures of alcohol‐denatured proteins have been studied using various techniques, including differential scanning calorimetry,20 circular dichroism (CD),21, 22 nuclear magnetic resonance (NMR),4, 23 ultrasound,15 mass spectrophotometry,8 small‐angle X‐ray scattering,24, 25 and Fourier‐transform infrared spectroscopy (FTIR) 10. NMR, FTIR, and CD can all be used to directly investigate the secondary structures, but CD spectroscopy is the most widely used because it is very sensitive to the local secondary structures and the CD spectra are measurable for any proteins at a low concentration under various solvent conditions.…”