Protein Engineering Protocols - Mycobacteriology research center

52 Mason et al.determination of the peptides. Crudely speaking, for the archetypal dimeric coiledcoils, such as GCN4 and Jun proteins, there is a preponderance of Lys, Asp, Arg,Glu, and Asn at these positions. Also present, albeit infrequently, is Ala, whichpresumably adds extra stability to the helix, perhaps in cases in which residues inclose spatial proximity have caused the overall α-helical propensity to lower.2.4.1. Helical LengthGenerally speaking, as the length of the coiled coil chain increases, a (nonlinear)increase in stability is observed (61). This is because the sequence of thecoiled coil will play an additional major role. For example, Lau and Hodges constructeda 29-mer with greater stability than tropomyosin, a 284-residue coiledcoil (see Note 25; ref. 62). One would expect the coiled coil structure to becomemore stable with length on the grounds of increased hydrophobic burial, hydrogenbonds, and polar interactions, and, again, this was proven to be the case in alength study of a designed homodimeric peptide (see Note 26; ref. 63), althougha minimum length of two heptads (see Note 27; ref. 64) is also required to permitassociation in the case of a homotrimer. The coiled coil domain of the Lacrepressor has been used as the basis to assess the effects of chain length on stabilityand folding (65). Unsurprisingly, the dissociation constant of the tetramersdecreased as the number of heptads in each helix increased (see Note 28).Somewhat more surprising was the fact that a tetramer with as few as four heptadsin each helix folded cooperatively, with no evidence for a dimeric intermediate.Long coiled coils, such as tropomyosin and myosin heavy-chain domain are,in contrast to short coiled coils, not enriched in the core exclusively with bulkynonpolar amino acids. Rather, because of the stability afforded from the lengthof the protein, such proteins contain clusters of small nonpolar or chargedresidues (66). These residues account for approx 40% of the core. Such destabilizingclusters consist mainly of Ala, because this residue is less destabilizingto the core than a polar amino acid (being easier to pack than a polar side chain),and is able to contribute favorably to the overall helical propensity. This meansthat although a stability increase proportional to the gain in heptads is not observedin these long coiled coils, from an evolutionary point of view, such a vast gain instability is not required. Instead, hydrophobic stabilizing clusters afford the coiledcoil the necessary stability, whereas destabilizing clusters do not, but do maintainthe helical structure. These clusters, predominant in Ala, increase the flexibility andlocal unfolding in such regions without affecting the overall stability of the coiledcoil, presumably allowing the protein to exercise its specific biological function.2.4.2. Helix PropensityLitowski and Hodges have reported that increasing the α-helical propensity ofnoncore residues by exchanging Ser for Ala (the amino acid of highest helical