Unfolding bovine α -lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations

Abstract

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.

Molecular Analytical Assessment of Thermally Precipitated α-Lactalbumin after Resolubilization

“…Another reason could be that the refolded α-la samples may contain several different conformations in one sample. Time-resolved IR studies suggest that the thermally unfolded apo-α-la likely initially transitions to a molten globule state, followed by either parallel or sequential folding pathways to resume the native holo-α-la state [ 41 ]. Though, a correct refolding to native state remains a challenging task, especially, when the protein was kept in a thermally unfolded state for a longer time and was further exposed to mechanical stress through stirring and centrifugation.…”

Resolving Dynamics in the Ensemble: Finding Paths through Intermediate States and Disordered Protein Structures

“…To leverage this general physical property, the temperature-jump (T-jump), usually induced by a laser pulse that excites the overtone stretch mode of water in aqueous solution, can trigger a temperature increase within nanoseconds that lasts up to milliseconds. 4,22,64 This indirect light activated pump−probe approach has been widely used to observe disordered protein regions and oligomerization mechanisms. 4,22 To utilize computational refinements for time-resolved processes requires both a time-resolved experimental input and MD trajectories to efficiently sample structures away from the stationary state.…”

“…4,22,64 This indirect light activated pump−probe approach has been widely used to observe disordered protein regions and oligomerization mechanisms. 4,22 To utilize computational refinements for time-resolved processes requires both a time-resolved experimental input and MD trajectories to efficiently sample structures away from the stationary state. In MD, the time-resolved structural changes are visible, but it is especially challenging to ensure sampled configurations are consistent with the experimental ensemble after a perturbation such as T-jump, pH-jump, or another stroboscopic triggering event.…”

“…Specifically, a disordered region in the secondary structure of the molten globule state was identified as an intermediate state on the pathway toward further unfolded states. 4 The combination of indirect pump−probe measurements and tailored MD simulations demonstrates a more deterministic way of illustrating temperature-dependent IDP behavior.…”

“…The set of accessible protein structures that perform physiological functions is now known to be much more heterogeneous than originally thought due to intrinsic disorder within the proteome. − Resolving ensembles of heterogeneous structures has been a major challenge in the protein community, despite the many achievements in structural characterization at thermodynamic equilibrium. However, in vivo , proteins are subjected to various environmental stimuli that drive structures away from equilibrium, such as changes in pH, temperature, or ion concentrations that can perturb or tune desired cellular functions. , Understanding protein structural dynamics in response to these environmental signals is paramount to elucidating functional mechanisms.…”