Contrasting Roles for Symmetrically Disposed b-Turns in the Folding of a Small Protein Hongdi Gu, David Kim and David Baker* Department of Biochemistry University of Washington Seattle, WA 98195, USA To investigate the role of turns in protein folding, we have characterized the effects of combinatorial and site-directed mutations in the two b- turns of peptostreptococcal protein L on folding thermodynamics and kinetics. Sequences of folded variants recovered from combinatorial libraries using a phage display selection method were considerably more variable in the second turn than in the ®rst turn. These combinatorial mutants as well as strategically placed point mutants in the two turns had a similar range of thermodynamic stabilities, but strikingly different folding kinetics. A glycine to alanine substitution in the second b-turn increased the rate of unfolding more than tenfold but had little effect on the rate of folding, while mutation of a symmetrically disposed glycine residue in the ®rst turn had little effect on unfolding but slowed the rate of folding nearly tenfold. These results demonstrate that the role of b- turns in protein folding is strongly context-dependent, and suggests that the ®rst turn is formed and the second turn disrupted in the folding tran- sition state. # 1997 Academic Press Limited Keywords: protein folding kinetics; protein L; b-turns; phage display; combinatorial mutagenesis *Corresponding author Introduction Do turns in proteins play an active role in guid- ing structure formation during folding, or do they merely serve as pliant linkers of secondary struc- tural elements? Combinational mutagenesis studies of several different proteins have shown that fold- ing can proceed despite substantial sequence per- turbations in turns (Brunet et al., 1993; Predki et al., 1996; Predki & Regan 1995; Ybe et al., 1996; Zhou et al., 1996). However, the effects of large sequence changes on folding kinetics have not yet been examined. Such kinetic studies are essential for determining whether turn formation is a critical step in folding. The 62 residue B1 immunoglobulin G (IgG) binding domain of peptostreptococcal protein L is an excellent system for investigating the role of turns in folding. The NMR solution structure (Wikstrom et al., 1993) consists of a four-stranded b-sheet packed against a single a-helix; the order of secondary structure elements is bbabb (Figure 1). The two b-turns are diametrically opposed and both contain a glycine residue, making them ideal for experimental comparison. Both the kinetics and thermodynamics of folding of a tryptophan-con- taining variant of protein L (hereinafter the pseudo wild-type protein is referred to as protein L) are ®t well by a simple two-state model (Scalley et al., 1997; Yi & Baker, 1996). A phage display selection method has been developed that allows for the retrieval of rare sequences that fold from large combinational libraries (Gu et al., 1995). The prin- ciple behind the selection is that the IgG binding activity of protein L requires that the protein be properly folded (Gu et al., 1995). Here, we used a combination of random muta- genesis, site-directed mutagenesis, and biophysical characterization to investigate the role of the two b-turns in the folding of protein L. Sequence per- turbations in the two turns have very different effects on folding; the formation of the ®rst turn appears to be part of the kinetic bottleneck in the folding reaction. The results have important impli- cations for the folding of protein L and for the role of turns in protein folding more generally. Results Phage display selection To obtain a broad overview of the role of the two b-turns in the folding of protein L, we began by selecting functional, folded variants of the pro- tein from phage libraries in which the two turns were independently randomized. Since our goal J. Mol. Biol. (1997) 274, 588±596 0022±2836/97/490588±09 $25.00/0/mb971374 # 1997 Academic Press Limited