Protein Engineering Protocols - Mycobacteriology research center

Design of Coiled Coil Structures 39Table 1Influence of GCN4-p1 Core Mutations on Oligomerization StateGeometry of sidePosition a–d chains at a–d Oligomerization state TamGCN4-p1 b β–γ Dimer 53°CI–L β–γ Dimer >100°C (77°C)I–I β–β Trimer >100°C (70°C)L–I γ–β Tetramer >100°C (94°C)V–I β–β — c 73°CL–V γ–β Trimers 81°CV–L β–γ Mixture of dimers and trimers 95°CL–L γ–γ Trimers >100°C (76°C)Adapted from ref. 16.a T mmeasurements were performed in 50 mM phosphate buffer, pH 7.0; 150 mM NaCl; and10 µM peptide. In parentheses are T mvalues measured in 3 M GuHCl.b The wild-type GCN4-p1 differs from the V–L mutant by an Asn pair at the central core a position,which ensures dimer formation (see Subheading 2.1.2.).c Species that could not be assigned.packing geometries are defined by the angle that the Cα–Cβ bond of the knobforms with the Cα–Cα vector at the base of the hole on a projection from theend of the coiled helices. For a parallel dimer, a knob in position a (in heptad i)fits into the hole in the other helix that is lined up in a clockwise sequence (iflooking into the hole) by residues in positions a′ i, d′ i, g′ i–1, and d′ i–1. Accordingly,a knob at d iis in the hole lined by d′ i, a′ i, e′ i, and a′ i+1(Fig. 1).Exhaustive analysis of different core mutants of the coiled coil of GCN4(a yeast homolog to the Jun transcription factor, sometimes referred to as GCN4-p1, see Note 1) revealed different packing geometries for different oligomerizationstates (Table 1; ref. 16). Comparison of the side-chain packing in the X-raystructure of the GCN4-p1 dimer and a designed tetrameric GCN4 mutantshowed that the local geometries of the a and d layers are reversed in the twostructures. Parallel knobs-into-holes packing was found at the a layer of thedimer and at the d layer of the tetramer. In contrast, perpendicular knobs-intoholespacking was observed at the d layer of the dimer and the a layer of thetetramer. A third class of knobs-into-holes interaction appeared at the a and dpositions of the parallel trimeric variant (17). In both layers, the Cα–Cβ bond ofeach knob makes an approx 60° angle with the Cα–Cβ vector at the base of the correspondinghole. This arrangement was termed “acute” knobs-into-holes packing.These different geometries account for a distinct preference of amino acidsfor a certain oligomerization state. The following list describes the outcome ofseveral experiments in which various amino acids were tested in the context ofstability and oligomerization specificity.