Abstract
Unlike native proteins that are amenable to structural analysis at atomic resolution, unfolded proteins occupy a manifold of dynamically interconverting structures. Defining the conformations of unfolded proteins is of significant interest and importance, for folding studies and for understanding the properties of intrinsically disordered proteins. Short chain protein fragments, i.e., oligopeptides, provide an excellent test-bed in efforts to define the conformational ensemble of unfolded chains. Oligomers of alanine in particular have been extensively studied as minimalist models of the intrinsic conformational preferences of the peptide backbone. Even short alanine peptides occupy an ensemble of substates that are distinguished by small free energy differences, so that the problem of quantifying the conformational preferences of the backbone remains a fundamental challenge in protein biophysics. Here, we demonstrate an integrated computational-experimental-Bayesian approach to quantify the conformational ensembles of the model trialanine peptide in water. In this approach, peptide conformational substates are first determined objectively by clustering molecular dynamics snapshots based on both structural and dynamic information. Next, a set of spectroscopic data for each conformational substate is computed. Finally, a Bayesian statistical analysis of both experimentally measured spectroscopic data and computational results is carried out to provide a current best estimate of the substate population ensemble together with corresponding confidence intervals. This distribution of substates can be further systematically refined with additional high-quality experimental data and more accurate computational modeling. Using an experimental data set of NMR coupling constants, we have also applied this approach to characterize the conformation ensemble of trivaline in water.