Desolvation and development of specific hydrophobic core packing during Im7 folding

Abstract

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val-->Thr or Phe-->Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Phi-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (>2.5 kJ mol(-1)) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.

Fig. 1

Ribbon diagrams of Im7 (PDB code 1AYI) showing core residues mutated to probe (a) solvation and (b) overpacking. Helix I is coloured red, helix II yellow, helix III green and helix IV blue. The figure was created using UCSF Chimera.

Fig. 2

Folding and unfolding kinetics of the Im7 solvation variants. Chevron plots are shown in the left-hand panels (a, c, e and g); the corresponding initial and endpoint fluorescence data are shown in the right-hand panels (b, d, f and h). Variants created (a) in the N-terminal region and throughout helix I, (c) in helices II and III, and (e) in helix IV. Variants for which ΔΔG°_UN was too small to calculate Φ-values are shown in (g). To facilitate comparison, the fit to the wild-type Im7 data is shown as a black dotted line in all plots. All data were acquired at pH 7.0, 10 °C, in the presence of 0.4 M Na₂SO₄, and fitted to a three-state on-pathway model (see Materials and Methods). The resulting kinetic and thermodynamic parameters are shown in Table 1.

Fig. 3

(a) Fluorescence emission spectra of IV22T (red) and wild-type Im7 (black) in 0 M (continuous line) or 8 M urea (dotted line). (b) Equilibrium denaturation curve of IV22T followed by tryptophan fluorescence. The data are fitted to a two-state transition. 1D H NMR spectra of (c) IV22T and (d) wild-type Im7. All data were acquired at pH 7.0, 10 °C, in the presence of 0.4 M Na₂SO₄.

Fig. 4

Histogram displaying Φ-values for the Im7 solvation variants. Blue bars depict Φ_I; grey bars, Φ_TS2. Variants for which ΔΔG°_UN was too small to calculate Φ-values (< 2.5 kJ mol^− 1) are not shown. Errors bars depict the errors on each Φ-value propagated mathematically from the errors determined on the fit parameters (see Materials and Methods). Data for all the solvation variants are shown in Table 1.

Fig. 5

Comparison of the Φ-values measured for I and TS2 of Im7 using (a) solvation (Val→Thr, Phe→Tyr) or (b) truncation (Val→Ala, etc.) substitutions. Φ-values for the truncation variants were taken from Capaldi et al. Residues mutated are coloured according to their Φ-value (Table 1). Red indicates Φ < 0.4, green 0.4 < Φ < 0.7, blue Φ > 0.7. Data for Φ_I are shown in the left-hand panel and Φ_TS2 on the right.

Fig. 6

Fluorescence emission spectra of the overpacked Im7 variants in 0 M (continuous line) or 8 M (dotted line) urea. (a) Overpacked variants with fluorescence properties resembling that of the native wild-type protein; (b) variants that differ slightly in their fluorescence intensity compared with native wild-type Im7; (c) variants that have a fluorescence emission spectrum in 0 M urea that resembles that of the intermediate species (highly fluorescent, λ_max ∼ 335 nm). (d) Fluorescence emission spectra of Im9 and the overpacked Im9 variants created in this study.

Fig. 7

Folding and unfolding kinetics of overpacked Im7 and Im9 variants. Chevron plots are shown in the left-hand panels (a, c, e, g and i); the corresponding initial and endpoint fluorescence data are shown in the right-hand panels (b, d, f, h and j). Variants created in (a) the N-terminal region and the N-terminal half of helix I, (c) the C-terminal half of helix I, (e) helices II and III and (g) helix IV. To facilitate comparison, the fit to the wild-type Im7 data is shown as a black dotted line in all plots. All data were acquired at pH 7.0, 10 °C, in the presence of 0.4 M Na₂SO₄ and fitted to a three-state on-pathway model (see Materials and Methods). (i) Chevron plots for overpacked Im9 variants and (j) corresponding initial and endpoint fluorescence data. Im9 data were acquired at pH 7.0, 10 °C, and fitted to a two-state model (see Materials and Methods).

Fig. 8

Histogram displaying ΔΔG°_xy values for different states of the overpacked Im7 variants. Dotted black lines indicate ΔΔG°_xy ± 2.5 kJ mol^− 1.

Fig. 9

Schematic of Im7 folding. Helices are coloured as in Fig. 1. The β_T value for TS1 was taken from Ref. ; values for other states are taken from the wild-type Im7 data in Table 1. Water molecules are shown as black and green (^) symbols. While the core of TS1 is assumed to remain highly solvated, the data presented here demonstrate the substantial desolvation occurs as I forms. The final steps in folding involve the docking of core residues (Val42, Ile44 and L53) in the C-terminal portion of helix II and in helix III, which serve to lock the protein into its native structure. These key residues are highlighted on the ribbon diagram of native Im7 (PDB code 1AYI). Overpacking these positions thus prevents efficient folding to the native state such that I becomes partially or wholly populated at equilibrium.

Scheme 1

Reaction scheme for a three-state on-pathway folding mechanism.

Scheme 2

Reaction scheme for a two-state folding transition.