The protein folding process often proceeds through partially folded transient states. Therefore, a structural understanding of these disordered states is crucial for developing mechanistic models of the folding process. Characterization of unfolded states remains challenging due to their disordered nature, and incorporating multiple methods is necessary. Combining the time-resolved x-ray solution scattering (TRXSS) signal with molecular dynamics (MD), we are able to characterize transient partially folded states of bovine α-lactalbumin, a model system widely used for investigation of molten globule states, during its unfolding triggered by a temperature jump. We track the unfolding process between 20 µs and 70 ms and demonstrate that it passes through three distinct kinetic states. The scattering signals associated with these transient species are then analyzed with TRXSS constrained MD simulations to produce protein structures that are compatible with the input signals. Without utilizing any experimentally extracted kinetic information, the constrained MD simulation successfully drove the protein to an intermediate molten globule state; signals for two later disordered states are refined to terminal unfolded states. From our examination of the structural characteristics of these disordered states, we discuss the implications disordered states have on the folding process, especially on the folding pathway. Finally, we discuss the potential applications and limitations of this method.
Hsu, D. J., Leshchev, D., Kosheleva, I., Kohlstedt, K. L., and Chen, L. X. (2021)
Unfolding bovine α-lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations.
J. Chem. Phys. 154, 105101. (abstract)
The molten globule structures (cyan) sampled from the I state in the free energy basin defined in Fig. 4. Only the structures in the basin that are <1.5 kcal/mol are rendered. Each structure is aligned by Cα to the crystal structure (purple).