J. Mol. Biol. (1996) 257, 430–440 Structure of the Transition State for Folding of a Protein Derived from Experiment and Simulation Valerie Daggett 1 *, Aijun Li 1 , Laura S. Itzhaki 2 , Daniel E. Otzen 2 and Alan R. Fersht 2 * Independent experimental and theoretical studies of the unfolding of barley 1 Department of Medicinal chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive Chemistry, University of plausible three-dimensional structural models of the transition state. A Washington, Box 357610 very simple structure index is calculated along the sequence for the Seattle, WA 98195-7610 molecular dynamics-generated transition state models to facilitate USA comparison with the  F values. The two are in good agreement overall 2 MRC Unit for Protein (correlation coefficient = 0.87), which suggests that the theoretical models Function and Design should provide a structural framework for interpretation of the  F values. Cambridge Center for Protein Both experiment and simulation indicate that the transition state is a Engineering, University distorted form of the native state in which the -helix is weakened but Chemical Laboratory partially intact and the -sheet is quite disrupted. As inferred from the  F Lensfield Road, Cambridge values and observed directly in the simulations, the unfolding of CI2 is CB2 1EW, UK cooperative and there is a ‘‘folding core’’ comprising a patch on the -helix and a portion of the -sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived  values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three-dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting  F values and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.  1996 Academic Press Limited Keywords: molecular dynamics; protein engineering; transition state structure; folding pathways; folding nucleation *Corresponding authors Introduction An essential step in the experimental analysis of protein folding pathways is the determination of the structure of transition states (Fersht, 1995), which can only be accomplished through kinetic studies. A procedure has been described for analyzing transition state structure at the level of individual residues (Fersht, 1993, 1994). This analysis yields a parameter  F for the residue probed that is an index of local structure during folding. A  F value of 0 means, in terms of energy, that the interactions made by the residue of interest are completely lost in the transition state. Conversely, a  F value of 1 indicates that the interactions the residue makes in the native state are retained in the transition state. In the absence of artifacts,  F can be interpreted as reflecting the native structure formation in the transition state around the residue of interest. Intermediate values of  F signify that the structure is weakened because of either partial structure formation or a dynamic equilibrium between folded and unfolded conformations. These intermediate values are particularly difficult to interpret in structural terms, as the  F values and extent of native structure are not linearly related. Abbreviations used: CI2, chymotrypsin inhibitor 2; MD, molecular dynamics; NOE, nuclear Overhauser effect. 0022–2836/96/120430–11 $18.00/0  1996 Academic Press Limited