Formation of B2 (formed by H2, H3 and H4): (Fig. 3)
Formation of the B2 core was probed by six mutations: H2, W112A; H3, I126A, I129A, Y133A; and H4, R140A, S144A. The extent of helix formation was probed by 12 Ala to Gly mutations: H2, A113G, A114G, A117G; H3, A127G, A131G, A132G; and H4, A138G, A139G, A142G, A143G, A146G, A150G.
Helix 4 is the most structured region of the entire protein, with high Ala to Gly Φ-values at the N-terminal end, becoming lower towards the C-terminal end. The two B2 core residues in this helix both have high Φ-values; W112 (the only hydrophobic residue from H2 which packs into this core) has a medium Φ-value. All the Φ-values of helix 3 are close to zero; this helix plays no role in the formation of B2 in the TS. Thus, we infer that the B2 core is loosely structured in the TS with H2 packing onto H4.