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A unifying view has been recently proposed according to which the classical diffusion-collision and nucleation-condensation models may represent two extreme manifestations of an underlying common mechanism for the folding of small globular proteins. We report here the characterization of the folding process of the PDZ domain, a protein that recapitulates the three canonical steps involved in this unifying mechanism, namely: (i) the early formation of a weak nucleus that determines the native-like topology of a large portion of the structure, (ii) a global collapse of the entire polypeptide chain, and (iii) the consolidation of the remaining partially structured regions to achieve the native state conformation. These steps, which are clearly detectable in the PDZ domain investigated here, may be difficult to distinguish experimentally in other proteins, which would thus appear to follow one of the two limiting mechanisms. The analysis of the (un)folding kinetics for other three-state proteins (when available) appears consistent with the predictions ensuing from this unifying mechanism, thus providing a powerful validation of its general nature. molecular dynamics ͉ protein engineering ͉ transition state

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We exploit the availability of recent experimental data on a variety of proteins to develop a Web-based prediction algorithm (BPPred) to calculate several biophysical parameters commonly used to describe the folding process. These parameters include the equilibrium m-values, the length of proteins, and the changes upon unfolding in the solvent-accessible surface area, in the heat capacity, and in the radius of gyration. We also show that the knowledge of any one of these quantities allows an estimate of the others to be obtained, and describe the confidence limits with which these estimations can be made. Furthermore, we discuss how the kinetic m-values, or the Beta Tanford values, may provide an estimate of the solvent-accessible surface area and the radius of gyration of the transition state for protein folding. Taken together, these results suggest that BPPred should represent a valuable tool for interpreting experimental measurements, as well as the results of molecular dynamics simulations.Keywords: protein denaturation; urea; guanidine hydrochloride; guanidinium chloride; protein folding; m-values; SASA; radius of gyration; heat capacity; transition state; unfolded state; denatured state The possibility of interpreting quantities readily measurable experimentally in terms of descriptors of protein strucure has contributed very significantly to our understanding of the folding process. In a seminal work, Myers et al. (1995) considered an earlier suggestion by Schellman (1978) and showed that the change in solvent-accessible surface area (DSASA) upon unfolding is related linearly to the experimental m D-N -value (Pace 1986), which describes how the stability nG D-N of the native state of a protein decreases linearly with the concentration of denaturant (Tanford 1968(Tanford , 1970:The relationship between m-values and DSASA is extremely useful because it gives important insights into the determinants of protein stability and the equilibrium properties of proteins. In order to establish such a relationship, however, one needs an estimate of the value of SASA of the unfolded state. Despite recent advances (Mok et al. 2005), it is still very challenging to measure SASA directly in the denatured state. Myers et al. (1995) derived it from a tripeptide model (Shrake and Rupley 1973;

TI I27, a beta-sandwich domain from the human muscle protein titin, has been shown to fold via two alternative pathways, which correspond to a change in the folding mechanism. Under physiological conditions, TI I27 folds by a classical nucleation-condensation mechanism (diffuse transition state), whereas at extreme conditions of temperature and denaturant it switches to having a polarized transition state. We have used experimental Phi-values as restraints in ensemble-averaged molecular dynamics simulations to determine the ensembles of structures representing the two transition states. The comparison of these ensembles indicates that when native interactions are substantially weakened, a protein may still be able to fold if it can access an alternative transition state characterized by a much larger entropic contribution. Analysis of the probability distribution of Phi-values derived from ensemble averaged simulations, enables us to identify residues that form contacts in some members of the ensemble but not in others illustrating that many interactions present in transition states are not strictly required for the successful completion of the folding process.