Solution structure and dynamics of a de novo designed three-helix bundle protein

Abstract

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, alpha3D, is reported. The structure of alpha3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

Figure 1

Sequences of the α₃ family and of Coil-Ser. The residues are aligned with their corresponding heptad position in a coiled coil (26). α₃A through α₃C and Coil-Ser were chemically synthesized whereas α₃D was cloned and expressed in E. coli. The residues that are different between α₃D and α₃C are labeled in bold.

Figure 2

(a) NMR spectrum of α₃D (¹H,¹³C-CT–heteronuclear single-quantum coherence) illustrating the assignments of the methyl groups. Prochiral assignments of the methyl groups of valine and leucine were obtained by using a 10% ¹³C labeled sample (35). (b) Summary of the α₃D sequential NOEs. The size of the bar corresponds to the intensity of the NOE. The NOEs were taken from the ¹⁵N- and ¹³C-resolved nuclear Overhauser effect spectroscopy spectra. The three bond ³J_αN coupling constants determined for α₃D are displayed as filled circles and boxes corresponding to ³J_αN coupling constants <6.0 Hz or >8.0 Hz, respectively.

Figure 3

(a) Stereo diagrams of the 13 superimposed α₃D structures are shown with the hydrophobic core residues depicted in red and W4 and Y45 in yellow. The structures were aligned by using only the backbone atoms (residues 4–21, 24–45, and 51–70). The figure was generated by using the program molmol (58). (b) Stereo display of a ribbon diagram with the hydrophobic residues in red and W4 and Y45 in yellow of the lowest energy structure of α₃D. The figure was created by using the programs molscript (59) and raster3d (60).

Figure 4

(a) Protection factors for the main-chain amide protons of α₃D determined from hydrogen–deuterium exchange at 15°C, pH* 5.0 (in 50 mM deuterated acetate). The protection factors were calculated from the observed exchange rates and the intrinsic exchange rates (39). Absence of a bar indicates residues with amide protons exchanging in the dead time of the experiment (18 minutes) or a lack of a well resolved amide proton cross peak. (b) Assuming an EX2 exchange mechanism, the apparent free energy, ΔG_HX, was calculated from the protection factors, P_i, as RTln(P_i) (39). The dashed line represents the ΔG_u at 15°C determined calorimetrically by extrapolation of the Gibbs-Helmoltz equation, ΔG_T = ΔH_Tm (1 − T/T_m) + (T − T_m) ΔC_p − TΔC_pln(T/T_m). The shaded region denotes the estimated standard error in this parameter of ±0.5 kcal⋅mol⁻¹.