Using chimeric immunity proteins to explore the energy landscape for α-helical protein folding

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k_UN^H2O ≈ 2000 s^-1 at pH 7.0, 25°C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (β^TS 0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)^Im9, Im2 (1-64)^Im9 and Im2 (25-44)^Im9) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)^Im9 and Im2 (56-64)^Im9 fold in two-state transitions via transition states that are significantly less compact (β^TS ≈ 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (β^TS ≈ 0.9). Surprisingly, Im2 (30-64)^Im9 populates a highly collapsed species (β^I = 0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.