Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin

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...

Section: Summary and Discussioncontrasting

confidence: 57%

Section: Summary and Discussioncontrasting

confidence: 56%

Dissecting the mechanical unfolding of ubiquitin

Irbäck

Mitternacht

Mohanty

2005

“…Recent applications of this method to biologically related molecules have yielded novel structural and dynamical results not readily obtainable by other methods for small peptides (7,8), soluble helices (9, 10), membrane-bound helices (11), lipids (12), ␤-sheets (13,14), and model secondary structures (15,16). The 2D IR approach is one that can be immediately applicable to a wide variety of sample types ranging from solutions to solids, including aqueous and lipid environments.…”

mentioning

confidence: 99%

“…The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13 tertiary interaction ͉ vibrational spectra ͉ multidimensional spectroscopy T wo-dimensional IR spectroscopy (1-6) is a promising new method with which to probe structures and their motions in complex systems. Recent applications of this method to biologically related molecules have yielded novel structural and dynamical results not readily obtainable by other methods for small peptides (7,8), soluble helices (9, 10), membrane-bound helices (11), lipids (12), ␤-sheets (13, 14), and model secondary structures (15, 16).…”

mentioning

confidence: 99%

Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer

Fang

Senes

Cristian

et al. 2006

130

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13 tertiary interaction ͉ vibrational spectra ͉ multidimensional spectroscopy T wo-dimensional IR spectroscopy (1-6) is a promising new method with which to probe structures and their motions in complex systems. Recent applications of this method to biologically related molecules have yielded novel structural and dynamical results not readily obtainable by other methods for small peptides (7,8), soluble helices (9, 10), membrane-bound helices (11), lipids (12), ␤-sheets (13, 14), and model secondary structures (15, 16). The 2D IR approach is one that can be immediately applicable to a wide variety of sample types ranging from solutions to solids, including aqueous and lipid environments. The method exposes interactions between spatially nearby vibrational modes by converting three pulse photon echo signals into 2D spectral maps in the IR, analogous to 2D NMR spectra.The amide-I vibrational bands of polypeptides are highly degenerate because, for each residue, their frequencies are approximately the same and the interactions among them are not strong enough to separately display them as individual, assignable transitions. The combination of multiple isotope selection and 2D IR spectroscopy that spreads the transitions into two dimensions proves to be a useful strategy that can simplify and expose the underlying transitions of the diffuse amide bands and allow their features to be characterized at a residue level. For example the 2D IR spectrum of a water-soluble helix selectively substituted with 13 CA 16 O and 13 CA 18 O provided characteristics of pairs of coupled residues and the visualization of the sequence dependence of the structural dynamics (17). This paper introduces a comparable approach to investigate tertiary interactions between vibrational modes in Glycophorin A (GpA), a transmembrane (TM) dimer of interacting helices (Fig. 1). The GpA TM helix provides an excellent prototype for the study of TM helix association, a key event in the folding of membrane proteins (18). The dimer also is an appropriate model for these investigations, because it is stable in a variety of micelles, including SDS (19), and a solution NMR structure is available (20). There are two Gly residues at the dimer interface that allow an intimate contact of the main chain, a feature frequently observed in TM helical interfaces (21,22). Because vibrational coupling is sensitive to distance, the proximity of the backbones is advantageous. GpA also was selected because verification of the applicability of the 2D IR method to membrane protein aggregates is of great interest and because there is a chronic paucity o...

“…In this way, a ''movie'' of the molecular motion can be constructed (24). This experimental scheme has recently been realized to study optically triggered ␣-helix formation in a peptide with time-resolved 2D-IR spectroscopy (25) and to study thermally induced unfolding of a protein with dispersed vibrational echo spectroscopy (26). Applying such methods to study the motion of molecular devices, which, in many cases, can also be externally triggered (5,27), will reveal fundamental aspects of their physics and chemistry.…”

mentioning

confidence: 99%

Probing the structure of a rotaxane with two-dimensional infrared spectroscopy

Larsen

Bodis

Buma

et al. 2005

Femtosecond 2D-IR spectroscopy has been used to study the structure of a [2]rotaxane composed of a benzylic amide macrocycle that is mechanically interlocked onto a succinamide-based thread. Both the macrocycle and the thread contain carbonyl groups, and by determining the coupling between the stretching modes of these groups from the cross-peaks in the 2D-IR spectrum, the structure of the macrocycle-thread system has been probed. Our results demonstrate that 2D-IR spectroscopy can be used to observe structural changes in molecular devices on a picosecond time scale.femtosecond IR spectroscopy ͉ molecular machines I t has recently become possible to synthesize molecules that function like mechanical devices (1-4), such as switches (5-7), motors (8-11), brakes (12), turnstiles (13), and elevators (14). These manmade molecular machines can be considered as nanoscale versions of their macroscopic analogues. However, many well known macroscopic concepts no longer apply at a molecular level. For instance, the concept of viscous friction becomes meaningless as the size of moving object approaches that of the molecules of the surrounding medium and as the time scale of the motion approaches that of the molecules of this medium (15). This regime of device operation is a new one, where the elementary component motions (rotations around covalent bonds and the making and breaking of hydrogen bonds) and the fluctuations of the surroundings both take place on the picosecond or subpicosecond time scale. Hence, for a detailed understanding of the physics and chemistry of molecular devices, experiments that probe their motion on an ultrafast time scale are essential, and the insights obtained from such experiments should be important for potential applications.A promising technique for such experiments is 2D-IR spectroscopy. With 2D-IR spectroscopy, molecular conformations are determined by measuring couplings between molecular vibrations. The method was inspired by multidimensional NMR spectroscopy, in which couplings between nuclear spins are used for structure resolution (16). Like nuclear spins, normal modes are often well localized in the molecule (especially when they involve stretching of specific chemical bonds), and the coupling between them is mainly dipolar. The 2D-IR spectrum can therefore give direct access to the conformation of a molecule or its parts (17-23). The important advantage of 2D-IR spectroscopy as compared with 2D-NMR spectroscopy is its time resolution: The conformation can be determined with a time resolution determined by the free-induction decay of the transition, which is generally Ͻ1 ps. As a consequence, 2D-IR spectroscopy can be used to study motion in molecular systems on a time scale comparable to that of the elementary molecular motions. To this purpose, one triggers the motion (typically with a short optical pulse) and monitors the subsequent structural changes by recording 2D-IR spectra at different time delays with respect to the external trigger. In this way, a ''movie'' of the molecular...