Protein Engineering Protocols - Mycobacteriology research center

Design of Coiled Coil Structures 49usually have a complicated interaction pattern. These coiled coils have to fulfill anumber of criteria, such as biostability and extremely high specificity within a familyand almost no crossreactivity with coiled coils of other families (46).2.3. Helix OrientationThe majority of coiled coils fold into a parallel alignment, however, a growingnumber of structurally characterized proteins contain antiparallel coiled coildomains (47). Despite the growing recognition of the biological importance ofantiparallel coiled coils, the study of this class of molecules has been hamperedby the lack of well-behaved model systems. None of the short antiparallel coiledcoil domains found in proteins such as seryl–transfer RNA (tRNA) synthetaseor hepatitis delta-virus antigen have been shown to be sufficient for dimerizationwithout undergoing further self-association.Hodges and co-workers were the first to report the characterization of ade novo designed coiled coil that was constrained in an antiparallel orientationby an interior disulfide bond (48). This and other designed antiparallel coiledcoils were more stable than their respective parallel counterparts, with nearlyequivalent interhelical interactions (49,50). These data suggest that, assumingeverything is equal, the helix–dipole interactions (see Subheading 2.4.3.) favorthe antiparallel orientation.2.3.1. Core ResiduesThe core residues that pack against each other are a–a′ and d–d′ in parallelcoiled coils. Antiparallel coiled coils have a–d′ and d–a′ central packing, yieldingidentical packing layers (51).1. It has been shown that the relative position of Ala residues in the core of a de novodesigned coiled coil can control the parallel or antiparallel orientation (seeSubheading 2.1.1., item 3; ref. 52). By careful placement of the Ala in the middleheptad, either all-parallel or all-antiparallel tetramers are formed. This wasachieved by an alternating pair of Ala and Leu residues (Ala–Leu–Ala–Leu) ineach of the two planes at the central heptad core positions of the molecule. Such analternating core of small and large residues (Ala–Leu–Ala–Leu) is the best way toaccommodate these small side chains. In the parallel arrangement, the packingwould be all Ala in one plane and all Leu in the other plane and would, thus, result ina large cavity that would solvate the core and destabilize the molecule (see Note 19).2. In a similar experiment, the core Asn of the dimeric GCN4-p1 (see Note 2) wasexchanged to Ala. The result was an antiparallel trimer to avoid a core cavity (53).However, parallel trimers were obtained in the presence of benzene, which boundto the core cavity (54).3. In a recent design of an antiparallel homodimeric coiled coil, termed APH, stericmatching of β-branched (Ile at position d) and truncated (Ala at the opposinga′ position) side chains in the hydrophobic core were used, along with other features