TEMPLATE-ASSEMBLED SYNTHETIC PROTEINS WITH 4-HELIX-BUNDLE TOPOLOGY - TOTAL CHEMICAL SYNTHESIS AND CONFORMATIONAL STUDIES

The total chemical synthesis and conformational characterization of three template-assembled synthetic proteins or TASPs [designated T4-(4-alpha-11), T4-(4-alpha-15), and T4-(3-alpha-16,alpha-16')] are described. These TASPs were designed to adopt globular folded structures of four-helix-bundle topology. The four constituent helical building blocks of the putative four-helix bundles were attached to the epsilon-amino groups of a cyclic carrier molecule (template) of the formula Ac-CysLysAlaLysProGlyLysAlaLysCys-NH2, which serves to induce and direct intramolecular folding of the amphiphilic secondary structure elements. While T4-(4-alpha-11) and T4-(4-alpha-15) contain four identical helical building blocks, 11 (alpha-11) and 15(alpha-15) amino acid residues in length, T4-(3-alpha-16,alpha-16') incorporates two different sequences comprising 16 amino acid residues each attached to lysines 2, 4, 7, and 9 of the template, respectively. The TASPs were synthesized by solid-phase techniques and extensively purified by a combination of reversed-phase HPLC and high-performance ion-exchange chromatography. TASP molecules of very high purity could be obtained in this way, which demonstrates the capacity of solid-phase peptide synthesis for the rapid preparation of branched macromolecules of this size when used in combination with orthogonal analytical and purification techniques. Comparative conformational studies (isolated helical building blocks vs TASP) by means of CD and NMR spectroscopy in aqueous solution demonstrate that the template exerts a strong secondary structure stabilizing effect and suggest that T4-(4-alpha-15) as well as T4-(3-alpha-16,alpha-16') adopts the proposed four-helix-bundle structure. This is further supported by the high conformational stability of T4-(4-alpha-15) under denaturing conditions, the guanidine hydrochloride induced folding --> unfolding transition occurring at denaturant concentrations between 3 and 4 M. In contrast, T4-(4-alpha-11) exhibits a predominantly unordered conformation, indicating a critical chain length requirement for helix formation of > 11 amino acid residues in the template-induced folding process.