Protein Engineering Protocols - Mycobacteriology research center

54 Mason et al.the combined effects of intrinsic destabilization and unfavorable charge–helixdipole interaction with the negative pole of the helix dipole.2. During selection for heterodimeric coiled coils from two designed libraries withan equimolar mixture of Glu, Gln, Arg, and Lys residues at four e and four g positions(see Note 7), Arndt et al. observed an enrichment of negatively charged Gluand neutral Gln at the N-terminal part and positively charged Lys and Arg at theC-terminal part of selected coiled coil sequences (31). This bias is in good agreementwith the proposed charge–helix dipole interactions.2.4.4. Helix CappingA helix can be labeled as N′-Ncap-N1-N2-N3-N4-mid-C4-C3-C2-C1-Ccap-C′.Of these positions, N′, Ncap, Ccap, and C′ have nonhelical ψ and φ angles, andonly N1-N2-N3...C3-C2-C1 participate in the i → i + 4 hydrogen bonding thatis characteristic of the α-helix. The N1, N2, N3, C1, C2, and C3 residues areunique because their amide groups participate in i → i + 4 backbone–backbonehydrogen bonds using either only their CO (at the N terminus) or their NH (atthe C terminus) groups (see also Subheading 2.4.3.). The need for these groupsto form hydrogen bonds has powerful effects on helix structure and stability(74). From N4 (or C4) upward, the residues can satisfy both NH and OH backbonehydrogen bonds.1. In helix design, the most selective position for the stability at the N-terminus is theNcap position. The six best amino acids for this position are Ser, Asp, Thr, Asn,Gly, and Pro, with another 11 (Val, Ile, Phe, Ala, Lys, Leu, Tyr, Arg, Glu, Met, andGln) being strongly avoided (75). Of the six preferred residues for Ncap, Ser, Asp,and Thr are the best.2. A good example of an Ncap motif is Ser-Xaa-Xaa-Glu (Ncap-N1-N2-N3), in whicha reciprocal side chain/main chain interaction pattern (OH of the Ncap Ser to NH ofGlu, and the carboxyl group of Glu to NH of Ser) stabilizes the helix. Further stabilizationcan be achieved by hydrophobic residues before and after the Ser-Xaa-Xaa-Glu motif (76). Lu et al. introduced this capping motif in the GCN4 sequence andwere thereby able to stabilize the coiled coil by 1.2 kcal/mol (see Note 31; ref. 77).3. The N1 and C′ positions strongly favor Pro (it is sterically compatible because the proceedingresidues have nonhelical dihedral backbone angles), which is indeed a commonhelix termination motif, but should be avoided both in the main body of the helixand in the C3, C2, and Ccap positions. Pro, being the most water soluble of all aminoacids (78), is compatible with solvent-exposed positions at the helix ends, and alsorequires no hydrogen bonding acceptor because it lacks a backbone NH group (76).4. C-terminal capping motifs involve backbone–backbone hydrogen bonds, ratherthan the side chain to backbone hydrogen bonding that is observed between Ncapand N3. At the C-terminus backbone, hydrogen bonds are satisfied by posthelicalbackbone groups (e.g., C′ and the following C′′ in the Schellman motif (76). Thismeans that the C-terminus need only select for C′ residues that can adopt positiveφ angles, for example, Gly.