Protein Engineering Protocols - Mycobacteriology research center

46 Mason et al.insertions of nonstabilizing, nonhydrophobic core residues, which will selectagainst alternative structures.1. Sharma et al. designed a peptide (anti-APCp1) that is targeted to bind a coiled coilsequence from the adenomatous polyposis coli (APC) tumor suppressor protein,which is implicated in colorectal cancers (see Note 12; ref. 36). In this, they usedcore changes together with g/e′ interactions, rather than Asn pairings, to ascribespecificity to the interaction. They reasoned that the low requirement discoveredfor core residues in driving specificity is surprising considering their dominantinfluence on association, and that core mutations can have large effects on stabilityand specificity. To address this issue, they designed a peptide to bind to the first55 amino acids of APC (APC55) and mutated this anti-APCp1 to generate the morefrequentlyobserved a–a′ and d–d′ pairings based on covariation patterns at the aand d positions of keratin type I and type II heterodimers. They made three mutations(A41I at layer a and A2M and M44A at layer d) to change the wild-typeAla–Ala and Met–Met interactions to the more-frequently found Ala–Ile, Ala–Met,and Met–Ala interactions, respectively. Two further mutations (T6G in layer a andN30H in layer d) served to destabilize the respective homodimers with Gly–Gly andHis–His pairs. Additional e–g′ pairings optimized ionic interactions while directingagainst anti-APCp1 homodimerization. The resulting heterodimer, APCp1/APC55,was both stable and specific.2. Schnarr and Kennan formed heterotrimeric proteins by steric matching of corehydrophobic residues (37). In their study, unnatural residues of various side-chainlengths were used to promote specific heterotrimer formation. The authors replaceda core a position of GCN4 with Ala or cyclohexylalanine (see Note 13). The resultwas a sterically mismatched core layer in the trimer, with either three Ala or threecyclohexylalanines, generating steric void or repulsion, respectively. Two Ala and acyclohexylalanine, however, generated a heterotrimer with good steric matching. Theuse of nonnatural side chains can be used in this way to generate coiled coils. Theadditional bulk of the cyclohexylalanine complements the Ala core layers to providea steric match, whereas bulkier side chains only serve to destabilize the molecule.2.2.2. Polar Core ResiduesThe roles of polar core residues in directing specific oligomerization states ofcoiled coils were discussed in Subheading 2.1.2. Heterotypic core contacts thatpermit generation of heterospecificity in coiled coil pairings were mentioned inSubheading 2.1.1. Further specificity can be obtained by interaction of polarcore interactions with the outer residues.1. Next to Asn, Lys at position a is the most common buried polar residue in naturaldimeric coiled coils (19). Lys at position a can form an intrahelical electrostaticinteraction with an e position residue of the preceding heptad (27) as well as aninterhelical g′–a polar interaction with a g′ position polar residue of the precedingheptad of the opposing helix in a parallel dimer (26).