How force unfolding differs from chemical denaturation

Abstract

Single-molecule force spectroscopies are remarkable tools for studying protein folding and unfolding, but force unfolding explores protein configurations that are potentially very different from the ones traditionally explored in chemical or thermal denaturation. Understanding these differences is crucial because such configurations serve as starting points of folding studies, and thus can affect both the folding mechanism and the kinetics. Here we provide a detailed comparison of both chemically induced and force-induced unfolded state ensembles of ubiquitin based on extensive, all-atom simulations of the protein either extended by force or denatured by urea. As expected, the respective unfolded states are very different on a macromolecular scale, being fully extended under force with no contacts and partially extended in urea with many nonnative contacts. The amount of residual secondary structure also differs: A significant population of α-helices is found in chemically denatured configurations but such helices are absent under force, except at the lowest applied force of 30 pN where short helices form transiently. We see that typical-size helices are unstable above this force, and β-sheets cannot form. More surprisingly, we observe striking differences in the backbone dihedral angle distributions for the protein unfolded under force and the one unfolded by denaturant. A simple model based on the dialanine peptide is shown to not only provide an explanation for these striking differences but also illustrates how the force dependence of the protein dihedral angle distributions give rise to the worm-like chain behavior of the chain upon force. molecular dynamics simulations | atomic force microscopy | worm-like chain model T he protein folding problem, which has attracted a great deal of attention for the last 50 y, has greatly benefited from the interaction and the contribution of experiments, simulations, and theoretical approaches (1). Traditional, ensemble-averaged experimental techniques such as NMR, ϕ-value analysis, small-angle X-ray scattering, and many other spectroscopic techniques have provided key insights into the structural, thermodynamic, and kinetic aspects of protein folding (1, 2). More recently, advances both in time-resolved single-molecule techniques (3) and in computer simulations (1, 4) have considerably enriched our comprehension of the problem with unprecedented mechanistic details.A key player in the protein folding event is the unfolded state, which is the initial configuration of the protein before it folds. Because the folded configuration is often the most stable monomeric protein state under physiological conditions, generation of unfolded configurations requires the application of an external perturbation able to denature the protein. In general, this is achieved by raising the temperature (thermal denaturation), changing the pH, or by adding chemical denaturants such as urea (chemical denaturation). The protein unfolded state can then be studied at equilibrium (i.e., by maintain...

Probing the Molecular Origin of Native-State Flexibility in Repeat Proteins

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 This work thus sheds experimental light on the molecular basis for the elasticity of RPs in general and TPRs in particular, demonstrating that it can be modulated by weakening hydrophobic interactions at the interfaces between repeat units, therefore connecting single-molecule force spectroscopy and FRET experiments. It has been proposed that forced unfolding differs significantly from chemical denaturation due to the formation of very extended conformations in the former 48 . This difference is less relevant to the low force -low denaturant concentration regime discussed here, and indeed both force measurements and our own experiments lead to a similar range of values for the spring constants.…”

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes

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