Protein Engineering Protocols - Mycobacteriology research center

38 Mason et al.Understanding of the rules governing association of helices has already permittedthese proteins to be exploited in novel ways (11); for example, by fusingantibody Fv fragments to the helices to create a helix stabilized antibody (12),by fusing antibody scFv fragments to the helices to create miniantibodies (13),or as a thermo-sensor (in the case of TlpA, which was tagged to green fluorescentprotein, with fluorescence changes monitored as a result of structuralchanges in TlpA during temperature changes). This could permit measurementsof signal transduction processes involving coiled coil dimer formation (14).2. Materials and MethodsThe several different aspects comprising the specific design of coiled coils arediscussed in this section. We aim to facilitate the choice of amino acids at the coreand the edge positions to achieve the desired oligomerization state (Subheading2.1.), specificity (Subheading 2.2.), and helix orientation (Subheading 2.3.).Here, we also relate different design solutions to the respective stabilities. Thefourth section (Subheading 2.4.) relates to the overall stability, focusing onthe outer (b, c, and f) positions. In this chapter, we attempt to break down all ofthe aspects to be considered into the relevant subheadings. Nonetheless, by thevery nature of the interplaying factors leading to the formation of the coiled coil,discussions in these sections will invariably crossover in places. Subsections dealingwith different effects exerted by the same residue positions should, therefore,be regarded as companion pieces.2.1. Oligomeric StateAny protein must achieve the desired three-dimensional structure to functionproperly. Likewise, a coiled coil structure must adopt the correct oligomeric(quaternary) structure. In the following section, the main factors contributing tothe adoption of this correct state are discussed.2.1.1. The Core ResiduesThe a and d residues are called core residues because they form a hydrophobicstrip that winds around each helix. The nonpolar nature of the a and d repeatfacilitates oligomerization along one face of each helix. This is analogous to ahydrophobic core, which collapses during the folding of globular proteins, andrepresents a dominating contribution to the overall stability of the coiled coil.Consequently, the core residues exert a major influence on defining the oligomerizationstate.The hydrophobic side chains in positions a and d bury into the neighboringhelix in a “knobs-into-holes” manner, first described by Crick in 1953 (15). In thismodel, a side chain from one α-helix (the knob) packs into a space surroundedby four side chains of the opposite α-helix (the hole), and vice versa. These