Using Cooperatively Folded Peptides To Measure Interaction Energies and Conformational Propensities.

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The rates and equilibria of the folding of biopolymers are determined by the conformational preferences of the subunits that make up the sequence of the biopolymer and by the interactions that are formed in the folded state in aqueous solution. Because of the centrality of these processes to life, quantifying conformational propensities and interaction strengths is vitally important to understanding biology. In this Account, we describe our use of peptide model systems that fold cooperatively yet are small enough to be chemically synthesized to measure such quantities. The necessary measurements are made by perturbing an interaction or conformation of interest by mutation and measuring the difference between the folding free energies of the wild type (in which the interaction or conformation is undisturbed) and the mutant model peptides (in which the interaction has been eliminated or the conformational propensities modified). With the proper controls and provided that the peptide model system in question folds via a two-state process, these folding free energy differences can be accurate measures of interaction strengths or conformational propensities. This method has the advantage of having high sensitivity and high dynamic range because the energies of interest are coupled to folding free energies, which can be measured with precisions on the order of a few tenths of a kilocalorie by well-established biophysical methods, like chaotrope or thermal denaturation studies monitored by fluorescence or circular dichroism. In addition, because the model peptides can be chemically synthesized, the full arsenal of natural and unnatural amino acids can be used to tune perturbations to be as drastic or subtle as desired. This feature is particularly noteworthy because it enables the use of analytical tools developed for physical organic chemistry, especially linear free energy relationships, to decompose interaction energies into their component parts to obtain a deeper understanding of the forces that drive interactions in biopolymers. We have used this approach, primarily with the WW domain derived from the human Pin1 protein as our model system, to assess hydrogen bond strengths (especially those formed by backbone amides), the dependence of hydrogen bond strengths on the environment in which they form, β-turn propensities of both natural sequences and small molecule β-turn mimics, and the energetics of carbohydrate-protein interactions. In each case, the combination of synthetic accessibility, the ease of measuring folding energies, and the robustness of the structure of the Pin1 WW domain to mutation enabled us to obtain incisive measurements of quantities that have been challenging to measure by other methods.