Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Abstract

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.

Fig. 1.

FRET as a measure of intramolecular distances. (A) Structure of MNEI. The location of W4 and the residues (P97 and Q29) that were replaced by cysteine residues are shown. Both P97 and Q29 were mutated independently to C, to yield two different single cysteine-containing mutant proteins, Cys97 and Cys29, respectively. The structure was drawn from PDB file 1IV7 by using the program PyMOL (http://www.pymol.org). The sole thiol moiety in each protein was labeled with TNB (as described in SI Text) that quenches the fluorescence of W4 in a distance-dependent manner. The mutant proteins labeled with TNB are named as Cys97-TNB and Cys29-TNB. (B and C) Fluorescence emission spectra of unlabeled and TNB-labeled proteins. (B) Cys97 and Cys97-TNB; (C) Cys29 and Cys29-TNB. In both B and C, the solid blue line and the solid dark red line represent the fluorescence spectra of unlabeled and TNB-labeled native proteins, respectively. The dashed blue line and the dashed dark red line denote the fluorescence spectra of unlabeled and TNB-labeled unfolded protein, respectively. All of the fluorescence spectra in B and C were collected in an identical manner, with the excitation wavelength set to 295 nm.

Fig. 2.

Unfolding kinetics of the unlabeled proteins. The unfolding of (A, C, E, and G) Cys97 and (B, D, F, and H) Cys29 at pH 8 and 25 °C was monitored by change in: (A and B) far-UV CD signal at 222 nm; (C and D) near-UV CD signal at 270 nm; and (E and F) fluorescence signal of W4 at 340 nm. In A–F, the solid dark red line shows the kinetic trace of unfolding in 4 M GdnHCl, and the solid black line through the data is a fit to a single exponential equation. The black dashed line represents the signal of native protein. Insets compare the kinetic versus equilibrium amplitudes of unfolding. The dark red triangles represent the equilibrium unfolding transition, and the continuous line through the data represents a fit to a two-state N⇌U model. The blue open circles represent the t = ∞ signal, and the blue filled circles represent the t = 0 signal, respectively, obtained from fitting the kinetic traces of unfolding to a single exponential equation. The black dotted line is a linear extrapolation of the native protein baseline. (C and D Insets) The blue line through the t = 0 values of the kinetic unfolding traces represents a fit to a two-state N⇌ I model. (G and H) Comparison of the observed rate constants of unfolding as monitored by change in far-UV CD signal at 222 nm (circles), near-UV CD signal at 270 nm (blue inverted triangles), and fluorescence of W4 at 340 nm (dark red triangles). In G and H, the black lines through the data are a straight line fit to the average of the rate constants from the 3 probes. The error bars, wherever shown, represent the standard deviations of measurements from at least 3 separate experiments.

Fig. 3.

Kinetics of unfolding of unlabeled and TNB-labeled proteins as monitored by FRET. (A) Cys97-TNB; (B) Cys29-TNB. In A and B, the changes in FRET efficiency during unfolding in 4 M GdnHCl (dark red continuous curve) and 6 M GdnHCl (dark green continuous curve) are shown, and the dashed black line shows the FRET efficiency estimated in the native protein. The data in A and B were converted into D–A distances by using Eq. S2, and the distances are shown for (C) Cys97-TNB and (D) Cys29-TNB at 4 M GdnHCl (dark red continuous curve) and 6 M GdnHCl (dark green continuous curve). The black dashed line shows the D–A distance estimated in the native protein. Identical kinetics of unfolding were seen at both 15 μM and 50 μM protein concentrations, indicating the absence of any transient aggregation during the unfolding reaction.

Fig. 4.

Kinetic versus equilibrium amplitudes of the unfolding as monitored by FRET. (A) Cys97-TNB; (B) Cys29-TNB. In A and B, the changes in FRET efficiency during unfolding are shown. The data in A and B were converted into D–A distances by using Eq. S2, and the distances are shown in C and D for Cys97-TNB and Cys29-TNB, respectively. In A–D, the black circles represent the equilibrium unfolding data, and the black inverted triangles and the black triangles represent the t = 0 and the t = ∞ kinetic signals, respectively. The continuous black line represents a fit to a two-state N⇌U model, and the dotted black line is a fit of the native protein baseline and t = 0 kinetic data to a single exponential equation.