Real-time tracking of protein unfolding with time-resolved x-ray solution scattering

Abstract

The correct folding of proteins is of paramount importance for their function, and protein misfolding is believed to be the primary cause of a wide range of diseases. Protein folding has been investigated with time-averaged methods and time-resolved spectroscopy, but observing the structural dynamics of the unfolding process in real-time is challenging. Here, we demonstrate an approach to directly reveal the structural changes in the unfolding reaction. We use nano- to millisecond time-resolved x-ray solution scattering to probe the unfolding of apomyoglobin. The unfolding reaction was triggered using a temperature jump, which was induced by a nanosecond laser pulse. We demonstrate a new strategy to interpret time-resolved x-ray solution scattering data, which evaluates ensembles of structures obtained from molecular dynamics simulations. We find that apomyoglobin passes three states when unfolding, which we characterize as native, molten globule, and unfolded. The molten globule dominates the population under the conditions investigated herein, whereas native and unfolded structures primarily contribute before the laser jump and 30 μ s after it, respectively. The molten globule retains much of the native structure but shows a dynamic pattern of inter-residue contacts. Our study demonstrates a new strategy to directly observe structural changes over the cause of the unfolding reaction, providing time- and spatially resolved atomic details of the folding mechanism of globular proteins.

Tracking the ATP-binding response in adenylate kinase in real time

“…Time-resolved x-ray solution scattering (TR-XSS) experiments and subsequent structural refinement can track and visualize structural rearrangements in photoactive chemicals (37), protein dimerization (38), folding (39,40) and temperature-jump (41) processes, as well as large-scale conformational changes in soluble (42)(43)(44)(45) and membrane (46)(47)(48)(49) proteins. A TR-XSS approach has also been proposed to simultaneously track local and global structural rearrangements in a protein (50).…”

Scattering fingerprints of two-state dynamics

“…probed in Ref. [58][59][60]. A systematic study of the relaxation at various q may provide further details, which is, however, beyond the scope of the present proof-of-principle investigation.…”

“…from either of the states). A neat and experimentally interesting problem is the denaturant-driven unfolding [58,59] or folding [60] of proteins. To this end we analyze the irreversible unfolding/folding of the small 20-residue Trp-Cage protein construct TC5b into/from a Rouse chain (N = 20).…”

The three-dimensional structure of Drosophila melanogaster (6–4) photolyase at room temperature

“…Time-resolved solution scattering has provided structural insight into the release of the CTT in cryptochromes (Berntsson et al, 2019), in agreement with static SAXS structures of cryptochromes (Vaidya et al, 2013). The named techniques offer flexibility and time-resolution, and in some cases even structural insight (Takala et al, 2014(Takala et al, , 2016Henry et al, 2020;Ravishankar et al, 2020;Berntsson et al, 2017), but the structural specificity is usually limited. Timeresolved crystallography is able to reveal more specific information about how the protein responds to photoexcitation in photolyases and cryptochromes.…”