The dynamic nature of the folded and unfolded states of the villin headpiece helical subdomain

The symbiotic relationship between experiment and simulation is necessary for a complete understanding of biomolecular structure and dynamics. Computational approaches can provide structural populations and atomic detail, and describe motion not visible on the macroscopic level, which can be used to interpret the experimental average ensemble as well develop new experiments. In turn, simulations rely on experimental observables for validation of a particular model or method.;In this work, both tools are used collaboratively to study the structure of the folded and the unfolded states of proteins. Solution NMR and X-ray structures of the folded state are widely used as a reference for simulations and experiments. Recent work has shown that the denatured state contains structure that is important for understanding protein stability and the folding pathway. This knowledge can be utilized to understand and treat protein misfolding diseases.;One of the key model systems, the 36-residue villin headpiece helical subdomain (HP36), was chosen for these studies because of its simple topology, small size and fast iv folding properties. Structures of HP36 have been determined using X-ray crystallography and NMR spectroscopy, but the two structures exhibited clear differences. Molecular dynamics simulations and experimental double mutant cycles were used to show that the X-ray structure is the better representation of the folded state in solution.;Previous experimental evidence has suggested that there is residual structure in the denatured state of HP36. Fragment analysis has shown that the three individual helices of HP36 lack significant structure compared to a larger fragment containing the first two helices (HP21). These techniques, however, are low resolution and are unable to quantify low levels of helical structure and whether it occurs in the same regions as HP36. Simulations were used to quantify the structure in all of the fragments. The HP21 ensemble contains less helical structure than predicted by NMR experimental observables possibly due to deficiencies in sampling and the force field. To address these limitations, simulation methodology and models were investigated.