Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation

Abstract

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded beta-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28-37 residues), the availability of a quantitative reaction coordinate (phi(T)), the fast folding time scale (10s of micros), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.

Figure 1

Structure of the WT FBP/WW domain showing the main mutation sites and Trp-8. The ΔN/ΔC truncations are shown in red and blue, respectively. The sequence is summarized below the structure. This figure was created by using PYMOL (available at http://pymol.sourceforge.net/).

Figure 2

(A) Thermodynamic analysis based on CD (230 nm) of WT FBP WW domain, its mutants, and truncations. The truncated variants denature ≈10°C lower but have essentially the same cooperativity as WT and variants thereof. (B) Different spectroscopic probes yield superimposable transitions, the hallmark of thermodynamic two-state transitions. All other FBP variants tested behave similarly (data not shown).

Figure 3

(A) Relaxation kinetics of WT FBP WW domain below and above the transition midpoint. (Inset) Increase in the slow-phase amplitude as temperature is lowered, overlaid on the fraction of unfolded protein from Fig. 2A. (B) A Tyr11Arg mutation, even combined with an N-terminal truncation, does not alleviate the double-exponential kinetics. (C) Further modification of the construct in B by truncation of the four C-terminal residues restores apparent two-state behavior at all temperatures, whereas a Leu26Ala mutation in loop 2, which may enhance misregistration there, partially restores three-state behavior at low temperatures in the ΔNΔC Y11R variant background. (D) Trp30Phe and Trp30Ala mutations of the full-length protein fold in a single phase, which may be caused by a reporter effect, or by involvement of other C-terminal residues in the formation of an intermediate with nonnative structure.

Figure 4

(A) Folding and unfolding rate constants from temperature-jump experiments of ΔNΔC Y11R W30F. Excellent fits can be obtained to a quadratic expansion of the free energy with temperature; these fits are used to calculate free energies as a function of temperature. (B) Free energies derived from the rates in A and from the thermodynamic data in Fig. 2A. (C) Energetics of the ΔNΔC Y11R W30F WW domain, plotted as a function of φ_T from Eq. 3. The transition and native states are shown “unstressed” (40°C) and stressed (65°C).

Figure 5

Folding free-energy landscape of the FBP WW domain. The energy landscape is defined by two coordinates, the number of native contacts (q_native) and nonnative contact formation (q_C) involving loop 2/C terminus. The two dominant minima represent the ensemble of denatured states (D) and the native state (N). The third local minimum accounts for the third species (I) manifested in the folding of the WT FBP WW domain under conditions that favor the native state. The experimental results are best rationalized in terms of such a two-dimensional surface (see text).