Protein Engineering Protocols - Mycobacteriology research center

44 Mason et al.Table 2Systematic Change of the Central a and d Positions to Every Amino Acid Usinga Model Peptide That Otherwise Has a Val at the a and Leu at the d Positions aPosition aPosition dOligomerization b Normalized stability c Oligomerization Normalized stabilityLeu Trimer 100 (69% Dimer) 100Ile (61% Dimer) 105 Trimer 89Val (57% Trimer) 108 Trimer 63Tyr Trimer 74 Dimer 67Trp Dimer 55 (78% Dimer) 47Gln Trimer 41 (61% Dimer) 56Asn Dimer 56 (72% Trimer) 41Lys Dimer 37 Dimer 25Orn Dimer 10 Dimer 7Arg Dimer 31 Dimer 9His Trimer 28 (55% Dimer) 37Glu (54% Dimer) 10 Dimer 12Asp Dimer d — Dimer 24a Amino acids that lead to a defined oligomerization state are shown. (From refs. 32 and 33.)b Helices were disulfide bridged via an N-terminal Cys–Gly–Gly linker when assessing theoligomerization state (see Note 9). However, the reported oligomerization state relates to thenumber of helices.c Normalized stability represents the stability of each substituted analog relative to Gly = 0 andLeu = 100.d The Asp analog was 100% folded at 5ºC. At room temperature, the analog was only ~20% folded.2.1.3. Edge ResiduesThe e and g (edge) positions of the heptad repeat flank the a and d residues incoiled coil interfaces (Fig. 1). Burial of these positions highly depends on theoligomerization state. Consequently, the choice of amino acids at the e and g sitesmay be influenced by the oligomerization state. Table 3 shows the calculated percentburied surface area expressed as the fraction of accessible side-chain surfacearea in the isolated helix that becomes buried in the oligomer (16).1. Compared with the corresponding sites of dimers, the e and g positions of trimersare enriched for hydrophobic residues (Ile, Leu, Val, Phe, Tyr, and Trp) anddepleted of specific hydrophilic residues (Glu, Gln, Ser, and Arg; ref. 19). Thesepatterns are consistent with the extension of the hydrophobic interface of trimers,relative to that in dimers. This increase in percentage of hydrophobic residuescauses the width of the narrow hydrophobic face to increase, and, with it, the likelihoodof higher oligomerization states, in which more nonpolar burial can occurthan in a two helix coiled coil. This can be seen in Table 3, in which the percentageof hydrophobic burial at the e and g positions is increased by approx 40%.