A Desolvation Model for Trifluoroethanol-Induced Aggregation of Enhanced Green Fluorescent Protein

Abstract

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.

Folding of intrinsically disordered plant LEA proteins is driven by glycerol‐induced crowding and the presence of membranes

“…2,2,2‐trifluoroethanol (TFE) is the most commonly used cosolvent to investigate the propensity of proteins to fold into an α‐helical structure . Disordered proteins can undergo a gradual coil‐to‐helix transition as increasing amounts of TFE are added to the solution and this behavior was also observed by CD spectroscopy for the five investigated proteins (Fig. A–E).…”

Structural basis of fluorescence quenching in caspase activatable‐GFP

“…It is not surprising that GFP 1-10 is in an aggregated state, even the robust mature GFP can be coaxed to aggregate upon the addition of 2,2,2-trifluoroethanol (TFE), a chemical commonly used to induce aggregation in proteins. 30 The observation that CA-GFP is not as aggregated then supports the hypothesis that it is more ordered than GFP [1][2][3][4][5][6][7][8][9][10] .…”

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

“…Alcohols, particularly the fluorinated alcohol 2,2,2-trifluoroethanol (TFE), are perhaps the most used type of co-solvents to investigate protein aggregation and have been used in the past to study αS in vitro under shaking/stirring conditions. 30 , 31 Here we have used quiescent conditions and found that low concentrations of alcohols such as methanol (MeOH) or TFE (see Fig. S3 † for the analysis of other types of alcohols), increased significantly the rate of αS aggregation (100 μM αS in PBS buffer pH 7.4, 37 °C, in quiescence; these conditions have been used for all further aggregation experiments) and increasing concentrations of alcohols resulted in shortened aggregation lag times ( Fig.…”