Abstract
In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a “caging” strategy to trap the β-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 μs, derived from a structural rearrangement in β-sheet refolding. The event lasts 10 times longer than the timescale of β-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a β-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a β-sheet.
Highlights
Protein folding is an important issue in protein science
The zipper model suggests that turn formation is the first step in β-sheet folding, as it directs β-strand orientation[16, 20, 22,23,24,25,26], whereas the hydrophobic collapse model emphasizes the importance of hydrophobic interactions in proteins and the entropy increase of water molecules in driving protein folding[27], While most researches focus on the refolding/unfolding process of β-sheet peptides, the results from the β-sheet peptides are not necessarily the same as the folding behavior of the β-sheet within a protein, in which the interactions between the β-sheet and the neighboring residues can affect the process
Photocleavage of the cage was initiated by pulse laser, and the resulting volume change from the heat release/absorption in the cage release event and the consequent ubiquitin refolding events could be recorded by time-resolved photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD)[16, 28,29,30,31]
Summary
Protein folding is an important issue in protein science. Figure 1 depicts a protein folding/misfolding funnel model[1, 2]. To understand how proteins fold, one common approach is using site-directed mutagenesis to explore the role of individual residues in protein stability and folding kinetics[6,7,8]. Another common approach is using short synthetic peptides corresponding to the target structural segment to study how local secondary structure forms in the early stage of folding, which is situated at the top of the funnel model of protein folding (Fig. 1). We were able to solve the refolding process of ubiquitin from the misfolded state to the native state by combining the caging strategy, laser flash photolysis, PAC, and PBD
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