Using Chimeric Immunity Proteins to Explore the Energy Landscape for a-Helical Protein Folding Neil Ferguson 1 , Wei Li 2 , Andrew P. Capaldi 1 , Colin Kleanthous 2 and Sheena E. Radford 1 * 1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 2 School of Biological Sciences University of East Anglia Norwich NR4 7TJ, UK 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 H 2 O UN  2000 s 1 at pH 7.0, 25  C) in apparent two-state tran- sitions, through rate-limiting transition states that are highly compact (b 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 pro- teins the introduced sequence variation results in altered kinetic beha- viour. At low urea concentrations, Im2 (1-29) Im9 and Im2 (56-64) Im9 fold in two-state transitions via transition states that are signi®cantly less com- pact (b 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 com- pact species (b TS  0.9). Surprisingly, Im2 (30-64) Im9 populates a highly collapsed species (b I  0.87) in the dead-time (2.5 ms) of stopped ¯ow measurements. These data indicate that whilst topology may place signi®cant constraints on the folding process, speci®c inter-residue inter- actions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape. # 2001 Academic Press Keywords: chimera; intermediate; topology; sequence; transition state *Corresponding author Introduction Understanding how proteins fold to their native conformation is a formidable challenge. However, the increasing synergy between theory and exper- iment (Daggett et al., 1996; Mun Ä oz & Eaton, 1999; Riddle et al., 1999), suggests that resolving this complex problem may now be within reach. Key to this has been the use of detailed kinetic analyses, which has allowed some of the different states vis- ited during folding and unfolding to be character- ised. The protein engineering method (Fersht et al., 1991) has been particularly powerful in that it has allowed the transition states of proteins to be struc- turally characterised (Serrano et al., 1992; Itzhaki et al., 1995; No È lting et al., 1997; Fulton et al., 1999; Kragelund et al., 1999; Lorch et al., 1999; Mateu et al., 1999) and, in some cases, compared with those observed in proteins of similar topology (Villegas et al., 1998; Chiti et al., 1999; Martõ Ânez & Serrano, 1999; Riddle et al., 1999) or predicted by theoretical models (Fulton et al., 1999; Riddle et al., 1999). For most small single domain proteins studied thus far the rate-limiting transition state contains an array of partially formed interactions involving both local and non-local contacts. The observation that mutation of one or more residues (Serrano et al., 1992; Itzhaki et al., 1995; No È lting et al., 1997; Fulton et al., 1999; Kragelund et al., 1999; Lorch et al., 1999; Mateu et al., 1999), restrict- ing the range of amino acid types in directed pro- tein evolution (Riddle et al., 1997), or changing solvent conditions (Martõ Ânez & Serrano, 1999), does not radically change the transition states of most proteins suggests that global properties of the polypeptide chain are important in de®ning this Present address: N. Ferguson, Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK. E-mail address of the corresponding author: s.e.radford@leeds.ac.uk doi:10.1006/jmbi.2001.4492 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 393±405 0022-2836/01/010393±13 $35.00/0 # 2001 Academic Press