A Designed Well-Folded Monomeric Four-Helix Bundle Protein Prepared by Fmoc Solid-Phase Peptide Synthesis and Native Chemical Ligation

The design and total chemical synthesis of a monomeric native‐like four‐helix bundle protein is presented. The designed protein, GTD‐Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix‐loop‐helix GTD‐43. GTD‐Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide–thioester fragment was prepared by anchoring the free γ‐carboxy group of Fmoc‐Glu‐allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine–thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide–thioester. The structure of the synthetic protein was examined by far‐ and near‐UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native‐like monomeric protein consistent with the design. Heat‐induced unfolding was studied by tryptophan absorbance and far‐UV CD. The thermal unfolding of GTD‐Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl‐induced unfolding were evaluated by far‐UV CD monitoring and the unfolding exhibited a cooperative transition that is well‐fitted by a two‐state mechanism from the native to the unfolded state. GTD‐Lig clearly shows the characteristics of a native protein with a well‐defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins. A monomeric 90‐residue‐long native‐like four‐helix bundle protein (see picture) has been prepared by thiol chemical ligation and standard fluorenylmethoxycarbonyl (Fmoc) chemistry. Its design, total chemical synthesis, and structural characterization are described.