Prediction of the translocation kinetics of a protein from its mechanical properties

Proteins are actively unfolded to pass through narrow channels in macromolecular complexes that catalyze protein translocation and degradation. Catalyzed unfolding shares many features that characterize the mechanical unfolding of proteins using the atomic force microscope (AFM). However, simulations of unfolding induced by the AFM and when a protein is translocated through a pore suggest that each process occurs by distinct pathways. The link, if any, between each type of unfolding, therefore, is not known. We show that the mechanical unfolding energy landscape of a protein, obtained using an atomistic molecular model, can be used to predict both the relative mechanical strength of proteins when unfolded using the AFM and when unfolded by translocation into a pore. We thus link the two processes and show that the import rate through a pore not only depends on the location of the initiation tag but also on the mechanical properties of the protein when averaged over all the possible geometries that are relevant for a given translocation initiation site.     

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM,molecular dynamics simulations,NMR and protein engineering

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.     

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy,protein engineering and molecular dynamics simulations

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.     

The mechanical stability of ubiquitin is linkage dependent

Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C-linked and Lys48-C-linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.     

Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.     

Mechanical Network in Titin Immunoglobulin from Force Distribution Analysis

The role of mechanical force in cellular processes is increasingly revealed by single molecule experiments and simulations of force-induced transitions in proteins. How the applied force propagates within proteins determines their mechanical behavior yet remains largely unknown. We present a new method based on molecular dynamics simulations to disclose the distribution of strain in protein structures, here for the newly determined high-resolution crystal structure of I27, a titin immunoglobulin (IG) domain. We obtain a sparse, spatially connected, and highly anisotropic mechanical network. This allows us to detect load-bearing motifs composed of interstrand hydrogen bonds and hydrophobic core interactions, including parts distal to the site to which force was applied. The role of the force distribution pattern for mechanical stability is tested by in silico unfolding of I27 mutants. We then compare the observed force pattern to the sparse network of coevolved residues found in this family. We find a remarkable overlap, suggesting the force distribution to reflect constraints for the evolutionary design of mechanical resistance in the IG family. The force distribution analysis provides a molecular interpretation of coevolution and opens the road to the study of the mechanism of signal propagation in proteins in general.     

Mechanically unfolding the small, topologically simple protein L

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.     

Mechanical unfolding pathway and origin of mechanical stability of proteins of ubiquitin family: An investigation by steered molecular dynamics simulation

Like the muscle protein Titin, proteins of the ubiquitin family exhibit a parallel strand arrangement, but otherwise having a distinctly different fold and not involved in an obvious load‐bearing function, exhibit high resistance to mechanical unfolding. We have applied all‐atom molecular dynamics simulation technique in implicit solvent to present a deep insight into the force‐induced unfolding pathway of three proteins—ubiquitin, NEDD8, and SUMO‐2—all having almost similar structural features. Two intermediates evolve in the unfolding pathway of each of the three proteins. The first intermediate, which has already been identified in case of ubiquitin by earlier simulation results, is similar for ubiquitin and NEDD8, but different in SUMO‐2. We have found a new intermediate with β3–β4 hairpin and some residual α‐helical character; and this intermediate is common for all the three proteins. Thus, proteins of the ubiquitin family pass through a well‐defined conformation in their force‐induced unfolding pathway. Reason behind the higher mechanical stability of the proteins with parallel strand structures like Titin has also been identified. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin

Single-protein force experiments have relied on a molecular fingerprint based on tethering multiple single-protein domains in a polyprotein chain. However, correlations between these domains remain an issue in interpreting force spectroscopy data, particularly during protein folding. Here we first show that force-clamp spectroscopy is a sensitive technique that provides a molecular fingerprint based on the unfolding step size of four single-monomer proteins. We then measure the force-dependent unfolding rate kinetics of ubiquitin and I27 monomers and find a good agreement with the data obtained for the respective polyproteins over a wide range of forces, in support of the Markovian hypothesis. Moreover, with a large statistical ensemble at a single force, we show that ubiquitin monomers also exhibit a broad distribution of unfolding times as a signature of disorder in the folded protein landscape. Furthermore, we readily capture the folding trajectories of monomers that exhibit the same stages in folding observed for polyproteins, thus eliminating the possibility of entropic masking by other unfolded modules in the chain or domain-domain interactions. On average, the time to reach the I27 folded length increases with increasing quenching force at a rate similar to that of the polyproteins. Force-clamp spectroscopy at the single-monomer level reproduces the kinetics of unfolding and refolding measured using polyproteins, which proves that there is no mechanical effect of tethering proteins to one another in the case of ubiquitin and I27.     

Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin

Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein''s mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.     

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.     

Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force-extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 microm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules.     

Ligand Binding Mechanics of Maltose Binding Protein

In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.     

Single-molecule unfolding force distributions reveal a funnel-shaped energy landscape

The protein folding process is described as diffusion on a high-dimensional energy landscape. Experimental data showing details of the underlying energy surface are essential to understanding folding. So far in single-molecule mechanical unfolding experiments a simplified model assuming a force-independent transition state has been used to extract such information. Here we show that this so-called Bell model, although fitting well to force velocity data, fails to reproduce full unfolding force distributions. We show that by applying Kramers' diffusion model, we were able to reconstruct a detailed funnel-like curvature of the underlying energy landscape and establish full agreement with the data. We demonstrate that obtaining spatially resolved details of the unfolding energy landscape from mechanical single-molecule protein unfolding experiments requires models that go beyond the Bell model.     

Spectrin domains lose cooperativity in forced unfolding

Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-beta immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.     

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.     

The effect of core destabilization on the mechanical resistance of I27

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.