Experimental validation of the role of trifluoroethanol as a nanocrowder

Abstract

Trifluoroethanol (TFE) is commonly used to induce protein secondary structure, especially α-helix formation. Due to its amphiphilic nature, however, TFE can also self-associate to form micellelike, nanometer-sized clusters. Herein, we hypothesize that such clusters can act as nanocrowders to increase protein folding rates via the excluded volume effect. To test this hypothesis, we measure the conformational relaxation kinetics of an intrinsically disordered protein, the phosphorylated kinase inducible domain (pKID), which forms a helix-turn-helix in TFE solutions. We find that the conformational relaxation rate of pKID displays a rather complex dependence on TFE percentage (v/v): while it first decreases between 0 and 5%, between 5 and 15% the rate increases and then remains relatively unchanged between 15 and 30% and finally decreases again at higher percentages (i.e., 50%). This trend coincides with the fact that TFE clustering is maximized in the range of 15-30%, thus providing validation of our hypothesis. Another line of supporting evidence comes from the observation that the relaxation rate of a monomeric helical peptide, which due to its predominantly local interactions in the folded state is less affected by crowding, does not show a similar TFE dependence.

Figure 1

(A) CD spectra of pKID collected at 1 °C and in aqueous solutions of different TFE percentages, as indicated. (B) The corresponding CD T-melts of these samples at 222 nm.

Figure 2

Representative trace of the relaxation kinetics of the pKID peptide in a 30% TFE solution in response to a T-jump from 5.7 to 11 °C, probed at 1630 cm^–1. The smooth line represents the best fit of this curve to a single-exponential function with a time constant of 1.8 ± 0.1 μs.

Figure 3

Temperature dependence of the relaxation rate constant of pKID measured for different TFE solutions, as indicated. For easy comparison, the results are presented in two panels: (A) 0–15% TFE and (B) 15–50% TFE. The solid lines shown are to guide the eye.

Figure 4

Cartoon illustration of the effect of TFE on the folding and unfolding free energy barriers of pKID. In scenario A, ΔG^⧧_U,W > ΔG^⧧_U,TFE and ΔG^⧧_F,W = ΔG^⧧_F,TFE, whereas in scenario B ΔG^⧧_U,W = ΔG^⧧_U,TFE and ΔG^⧧_F,W < ΔG^⧧_F,TFE.

Figure 5

(A) CD spectra of LEA collected at 1 °C and in aqueous solutions of different TFE percentages, as indicated. (B) The corresponding CD T-melts of these samples at 222 nm.

Figure 6

A representative trace of the relaxation kinetics of the LEA peptide in a 40% TFE solution in response to a T-jump from 3.8 to 8.4 °C, probed at 1664 cm^–1. The smooth line represents the best fit of this curve to a single-exponential function with a time constant of 0.9 ± 0.1 μs.

Figure 7

Relaxation rate constants of LEA versus temperature for different TFE solutions, as indicated.