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) approximate to 2000 s(-1) at pH 7.0, 25 degreesC) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta (TS) 0.93 and 0.96, respectively). Whilst the folding and unfolding proper ties 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 (beta (TS) approximate to 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 (beta (TS) approximate to 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta (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. (C) 2001 Academic Press.