Protein Engineering Protocols - Mycobacteriology research center

Design of Coiled Coil Structures 45Table 3Percentage of Buried Surface Areas for GCN4-p1Dimer and p-LI TetramerPosition GCN4-p1 dimer GCN4-pLI tetramera 87 92b 0 10c 1 19d 87 99e 26 72f 0 0g 27 66From ref. 14.The decrease in oppositely charged g ito e′ i+1pairs in trimers (12%) comparedwith dimers (23%) is consistent with this (19).2. Fairman et al. mutated the C-terminal homotetrameric coiled coil domain of thelac repressor to generate a heterotetramer (35). Peptides containing either all Lysor all Glu at the b and c positions, which flank the e and g positions weakly associate,but, if these are mixed, a highly stable tetramer is formed (see Note 11). Thisdemonstrates that, at least for tetramers, the b and c residues also play a significantrole in the stability of the coiled coil. This is a role akin to the g/e′ ionic interactionsfound in dimeric coiled coils, but with the widened hydrophobic interfaceof the tetramer extending to these residues, the ionic role falls further outwardsfrom the core to the b and c residues. By changing pH and salt levels, these ionpair interactions between Glu and Lys were shown to be responsible for theincreased stability. Additionally, these charges direct against homo-oligomers, andit may be that this unfavorable charge repulsion in potential homodimeric interactionsdrives the heterodimer formation (9,35).2.2. Pairing SpecificityThe following section discusses the importance of pairing specificity, andsome of the ways in which the coiled coil ensures that no other energeticallyfavorable structures can be accessed. Remarkably, despite their similarity insequence and structure, coiled coils interact preferentially with functional partners.This section analyzes the factors mediating such high selectivity.2.2.1. Core ResiduesThe patterning of hydrophobic residues (mostly Leu, Ile, and Val), as outlinedin Subheading 2.1.1., is a dominant driving force behind the associationof the helices. However, for this pattern to be observed so frequently, how canthe coils, at the same time, use the core to direct against alternative structuresforming? The answer is a complicated picture involving subtle changes, such as