Protein Engineering Protocols - Mycobacteriology research center

158 Bradley et al.Fig. 3. The design template for the initial four-helix bundle library. Nonpolar positions() were encoded by the degenerate NTN codon, and polar positions () wereencoded by the degenerate NAN codon. The defined residue positions were located atthe N- and C-terminal regions as well as the interhelical turn regions of the designedprotein.2.1.1. Binary Patterned Regions2.1.1.1. α-HELICAL DESIGNSBinary patterning exploits the periodicities inherent in secondary structures.α-Helices have a repeating periodicity of 3.6 residues per turn (Fig. 1A). To designan amphipathic segment of α-helical secondary structure, a binary pattern ofP-N-P-P-N-N-P (P represents polar and N, nonpolar) is used. Our initial α-helicaldesign focused on the four-helix bundle motif (Fig. 3). In this structure, thehydrophobic face of each of the helices is oriented toward the central core of thebundle, whereas the hydrophilic faces of the helices are exposed to the aqueousenvironment. The P-N-P-P-N-N-P pattern favors the formation of an amphiphilicα-helical secondary structure that can bury all nonpolar amino acids after formationof the desired tertiary structure. From our designed four-helix bundle libraries,more than 50 proteins have been purified and characterized. All have shown typicalα-helical circular dichroism spectra (see Note 1). Moreover, the collectionyielded several proteins with native-like properties, such as nuclear magnetic resonancechemical shift dispersion, cooperative chemical and thermal denaturations,and slow hydrogen/deutorium exchange rates (14–18).