A three-state mechanism for trifluoroethanol denaturation of an intrinsically disordered protein (IDP).

Relating the amino acid composition and sequence to chain folding and binding preferences of intrinsically disordered proteins (IDPs) has emerged as a huge challenge. While globular proteins have respective 3D structures that are unique to their individual functions, IDPs violate this structure-function paradigm because rather than having a well-defined structure an ensemble of rapidly interconverting disordered structures characterize an IDP. This work measures 2,2,2-trifluoroethanol (TFE)-induced equilibrium transitions of an IDP called AtPP16-1 (Arabidopsis thaliana phloem protein type 16-1) by using fluorescence, circular dichroism, infrared and nuclear magnetic resonance (NMR) methods at pH 4, 298 K. Low TFE reversibly removes the tertiary structure to produce an ensemble of obligate intermediate ($\mathrm{I}$) retaining the native-state ($\mathrm{N}$) secondary structure. The intermediate $\mathrm{I}$ is preceded by a non-obligate tryptophan-specific intermediate ${\mathrm{I}}_{\mathrm{w}}$ whose population is detectable for AtPP16-1 specifically. Accumulation of such non-obligate intermediates is discriminated according to the sequence composition of the protein. In all cases, however, a tertiary structure-unfolded general obligate intermediate $\mathrm{I}$ is indispensable. The $\mathrm{I}$ ensemble has higher helical propensity conducive to the acquisition of an exceedingly large level of α-helices by a reversible denaturation transition of $\mathrm{I}$ to the denatured state $\mathrm{D}$ as the TFE level is increased. Strikingly, it is the same $\mathrm{N}\rightleftharpoons \mathrm{I}\rightleftharpoons \mathrm{D}$ scheme typifying the TFE transitions of globular proteins. The high-energy state $\mathrm{I}$ characterized by increased helical propensity is called a universal intermediate encountered in both genera of globular and disordered proteins. Neither $\mathrm{I}$ nor $\mathrm{D}$ strictly show molten globule (MG)-like properties, dismissing the belief that TFE promotes MGs.

A surprisingly simple three-state generic process for reversible protein denaturation by trifluoroethanol.

Despite the rich knowledge of the influence of 2,2,2-trifluoroethanol (TFE) on the structure and conformation of peptides and proteins, the mode(s) of TFE-protein interactions and the mechanism by which TFE reversibly denatures a globular protein remain elusive. This study systematically examines TFE-induced equilibrium transition curves for six paradigmatic globular proteins by using basic fluorescence and circular dichroism measurements under neutral pH conditions. The results are remarkably simple. Low TFE invariably unfolds the tertiary structure of all proteins to produce the obligate intermediate (I) which retains nearly all of native-state secondary structure, but enables the formation of extra α-helices as the level of TFE is raised higher. Inspection of the transitions at once reveals that the tertiary structure unfolding is always a distinct process, necessitating the inclusion of at least one obligate intermediate in the TFE-induced protein denaturation. It appears that the intermediate in the minimal unfolding mechanism N⇌I⇌D somehow acquires higher α-helical propensity to generate α-helices in excess of that in the native state to produce the denatured state (D), also called the TFE state. The low TFE-populated intermediate I may be called a universal intermediate by virtue of its α-helical propensity. Contrary to many earlier suggestions, this study dismisses molten globule (MG)-like attribute of I or D.

Complete observation of all structural, conformational, and fibrillation transitions of monomeric globular proteins at submicellar sodium dodecyl sulfate concentrations.

Although considerable information is available regarding protein-sodium dodecyl sulfate (SDS) interactions, it is still unclear as to how much SDS is needed to denature proteins. The role of protein charge and micellar surfactant concentration on amyloid fibrillation is also unclear. This study reports on equilibrium measurements of SDS interaction with six model proteins and analyzes the results to obtain a general understanding of conformational breakdown, reorganization and restructuring of secondary structure, and entry into the amyloid fibrillar state. Significantly, all of these responses are entirely resolved at much lower than the critical micellar concentration (CMC) of SDS. Electrostatic interaction of the dodecyl sulfate anion (DS- ) with positive surface potential on the protein can completely unfold both secondary and tertiary structures, which is followed by protein chain restructuration to α-helices. All SDS-denatured proteins contain more α-helices than the corresponding native state. SDS interaction stochastically drives proteins to the aggregated fibrillar state.