A protein’s function is determined by its structure; plus, intrinsic disorder and flexibility gives rise to increasingly more functional conformations, which were previously thought to be irrelevant. One model for intrinsically disordered proteins is the 20-residue Trp-cage synthetic mini-protein. It is one of the smallest systems to exhibit a stable secondary structure and fast-folding dynamics less than 4 µs. These properties make it suitable for detailed theoretical investigations of protein folding, which have been performed over past decades. While various works based on molecular dynamics simulations predicted complex folding behavior for Trp-cage, these models have not been confirmed experimentally. Few spectroscopic works have inferred a single stable intermediate conformation on a pathway from folded to unfolded basins; however, no structural information is available for this intermediate folding step. To bridge the understanding of secondary structure dynamics of Trp-cage on nanosecond- to microsecond-timescales, we use time-resolved X-ray solution scattering (TRXSS) to probe the temperature-induced unfolding of Trp-cage. Laser-initiated environmental perturbations coupled with X-ray scattering allow for the observation of transient structural dynamics during the folding/unfolding process. Our results indicate the formation of an unfolded intermediate on the timescale of 1 μs, which undergoes complete unfolding within 5 μs. The ongoing structural analysis paves the way for direct benchmarking of theoretical models of protein folding produced with molecular dynamics simulations, which in turn will provide deeper insight into the process of secondary structure formation.

Determining the transient structures of a fast-folding mini-protein

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