Dissecting the mechanical unfolding of ubiquitin

Abstract

The unfolding behavior of ubiquitin under the influence of a stretching force recently was investigated experimentally by single-molecule constant-force methods. Many observed unfolding traces had a simple two-state character, whereas others showed clear evidence of intermediate states. Here, we use Monte Carlo simulations to investigate the force-induced unfolding of ubiquitin at the atomic level. In agreement with experimental data, we find that the unfolding process can occur either in a single step or through intermediate states. In addition to this randomness, we find that many quantities, such as the frequency of occurrence of intermediates, show a clear systematic dependence on the strength of the applied force. Despite this diversity, one common feature can be identified in the simulated unfolding events, which is the order in which the secondary-structure elements break. This order is the same in two-and three-state events and at the different forces studied. The observed order remains to be verified experimentally but appears physically reasonable. all-atom model ͉ force-induced unfolding ͉ Monte Carlo simulation T he 76-residue protein ubiquitin fulfills many important regulatory functions in eukaryotic cells through its covalent attachment to other proteins (1, 2). In many cases, the ubiquitin tag consists of a chain of ubiquitin domains (polyubiquitin), which is formed by linkages between an exposed lysine side chain of the last ubiquitin of a growing chain and the C terminus of a new ubiquitin. The fate of a polyubiquitin-tagged protein depends on the linkage. For example, Lys-48-C-linked polyubiquitin marks the protein substrate for proteasomal degradation (3).Recently, Fernandez and coworkers (4-7) and Chyan et al. (8) investigated the mechanical properties of polyubiquitin by single-molecule force spectroscopy. It was shown that Lys-48-Clinked as well as end-to-end (N-C)-linked polyubiquitin can withstand a stretching force; the average unfolding force was 85 pN for Lys-48-C linkage and Ϸ200 pN for N-C linkage (4). In these experiments, the polyubiquitin chains were pulled with a constant velocity. In another experiment on N-C-linked polyubiquitin, the stretching force was kept constant (6). At constant force, the fraction of unfolded ubiquitin domains was found to show an approximately single-exponential time dependence, as expected if the unfolding of individual domains is a simple Markovian two-state process. Nevertheless, the unfolding of individual domains sometimes occurred through intermediate states. The precise nature of these different unfolding pathways, including the structure of the intermediate states, remains to be determined. We stress that these intermediates are states along forced unfolding trajectories. To what extent there are significant folding intermediates for small proteins is a debated (9, 10), but different, issue. Here, we use Monte Carlo (MC) simulations to examine the unfolding of ubiquitin under a constant stretching force in atomic detail. Our calculations are b...

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

“…Unlike classical unfolding experiments using chemicals or large temperature jumps, the mechanical unfolding of proteins is a highly localized process mainly involving the rupture of a few key hydrogen bonds within the structure of the protein native state, which constitutes the crucial structural motif that provides the protein with mechanical stability (41). In the case of the ubiquitin protein, the mechanical clamp is placed between the ␤1-␤5 sheets (42). After the rupture of this mechanical clamp, which constitutes the major energy barrier for unfolding, the protein extends up to almost its contour length without any extra enthalpic energy cost.…”

Water’s role in the force-induced unfolding of ubiquitin

“…In atomic force spectroscopic studies of the elastomeric protein ubiquitin, the β-strands 1-5 serve as the force clamp under shear loading. Steered molecular dynamics (SMD) simulations (2, 4) showed that the shear loading force needs to break all four backbone hydrogen bonds (bbHBs) between the parallel β-strands 1-5 of ubiquitin in order to trigger protein unfolding (1,(5)(6)(7)(8). In SMD simulations of I27, Lu and Schulten observed that in the early stages of the pulling process individual bbHBs occasionally break and quickly reform, due to thermal fluctuations.…”

Protein Folding, Unfolding and Aggregation Studied Using an All-Atom Model with~a~Simplified Interaction Potential

“…The same model has also been used to study the mechanical unfolding of ubiquitin [16,17], a protein with 76 amino acids. The unfolding behavior of Fig.…”

“…The simulations of the mechanical unfolding of ubiquitin were carried out at constant force [16]. As in the constant-force experiments [72], the forces acted on the chain ends.…”

“…Without changing any parameters, the model was also used to study the aggregation properties of the 7-amino acid fragment Aβ 16−22 of the amyloid-β peptide associated with Alzheimer's disease, with very promising results [15]. Recently, the mechanical [16] and the thermal [17] unfolding of ubiquitin, a protein with 76 amino acids, was investigated, again using exactly the same model. All the simulations of this model were performed using Monte Carlo methods.…”