Mechanical Unfolding Pathway Research Articles

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods, may have important implications in drug development and discovery. Here, we present the first approach that employs computational High-Throughput Virtual Screening (HTVS) and Molecular Docking to search for small-molecules in chemical libraries that function as mechano-regulators of the stability of human CD4, receptor of HIV-1. Using single-molecule force spectroscopy we prove that these small-molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, i.e., mechano-regulation, paving the way towards mechanopharmacology.

Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

The Effect of Chain Connectivity on the Thermodynamic, Kinetic and Mechanical Properties of Azurin

Azurin, a blue-copper protein, is responsible for electron transfer in Pseudomonas aeruginosa. It has widely been studied for its stability, electronic and electron transport properties, with potential applications in biomaterials-based devices. The amino acid chain connectivity is of fundamental importance in modulating the protein stability and folding. Hence, here we studied the thermodynamic, kinetic and mechanical properties of a circular permutant of apo-azurin (Azu-CPN42), with its N-terminus at Asn42, and compared them with those of the wild-type apo-azurin (WT-Azu). While the spectroscopic and copper-binding properties of Azu-CPN42 are unaltered, we observe changes in protein stability. Equilibrium unfolding studies using GdHCl suggest a drop of ∼0.6M in Cm and ∼13 kJ/mol in free energy of unfolding for Azu-CPN42 compared to that of WT-Azu. Kinetic studies reveal increase in the spontaneous unfolding rate by two-orders of magnitude, while the spontaneous folding rate being similar. Mechanical properties probed using single-molecule atomic force microscope suggest parallel unfolding pathways via 2- and 3-states, similar to that of WT-Azu reported previously. Interestingly, the intermediate stability is lower than that of the native state of Azu-CPN42 by ∼10pN, in accord with that of WT-Azu. As anticipated, the mechanical unfolding pathways of Azu-CPN42 and WT-Azu are different, as the two proteins are pulled along different N-C termini. This resulted in different mechanical intermediates, with a broad distribution of unfolding contour lengths (∼3-25 nm) for Azu-CPN42 while those of WT-Azu are centered at 7.5 ± 1.5 nm. This huge diversity may arise due to mechanical unraveling from both C- and N- termini as opposed to only C-terminal unraveling observed in WT-Azu. Our study reveals a decreased thermodynamic and kinetic stability and altered mechanical unfolding pathway of azurin upon changing its chain connectivity through circular permutation.

Atomic Force Microscopy as a Powerful Multifunctional Tool for Probing the Behaviors of Single Proteins.

Proteins play a crucial role in regulating life activities and therefore investigating the behaviors of proteins is of critical significance for understanding the underlying mechanisms guiding the physiological and pathological processes of living organisms. Traditional molecular biochemical methods for protein assays are based on ensemble measurements which reflect the averaged behaviors of the whole molecular populations, veiling the rare activities of small molecular subpopulations. Achievements obtained in the past decades have proved that atomic force microscopy (AFM) is a powerful multifunctional tool for characterizing the behaviors of single proteins at work in aqueous conditions with unprecedented spatiotemporal resolution, which provides novel insights into nanoscopic molecular mechanisms and contributes much to the communities of protein biology. In this article, we review the recent advances in AFM-based single-protein analysis from several facets (including morphological imaging, single-molecule force recognition, mechanical unfolding pathways, and high-speed AFM molecular detection), and provide perspectives for future progression.

Mechanical and Structural Tuning of Reversible Hydrogen Bonding in Interlocked Calixarene Nanocapsules.

We present force probe molecular dynamics simulations of dimers of interlocked calixarene nanocapsules and study the impact of structural details and solvent properties on the mechanical unfolding pathways. The system consists of two calixarene "cups" that form a catenane structure via interlocked aliphatic loops of tunable length. The dimer shows reversible rebinding, and the kinetics of the system can be understood in terms of a two-state model for shorter loops (≤14 CH2 units) and a three-state model for longer loops (≥15 CH2 units). The various conformational states of the dimer are stabilized by networks of hydrogen bonds, the mechanical susceptibility of which can be altered by changing the polarity and proticity of the solvent. The variation of the loop length and the solvent properties in combination with changes in the pulling protocol allows to tune the reversibility of the conformational transitions.

Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the focus of many studies for investigating interdomain interactions within the native state and during folding. Previous studies used traditional biophysical methods (such as circular dichroism, tryptophan fluorescence, and NMR) to measure signals over a large ensemble of molecules, which made it difficult to observe transient changes in stability or structure during unfolding and refolding of single molecules. Here, we unfold single molecules of PGK using atomic force spectroscopy and steered molecular dynamic computer simulations to examine the conformational dynamics of PGK during its unfolding process. Our results show that after the initial forced separation of its domains, yeast PGK (yPGK) does not follow a single mechanical unfolding pathway; instead, it stochastically follows two distinct pathways: unfolding from the N-terminal domain or unfolding from the C-terminal domain. The truncated yPGK N-terminal domain unfolds via a transient intermediate, whereas the structurally similar isolated C-terminal domain has no detectable intermediates throughout its mechanical unfolding process. The N-terminal domain in the full-length yPGK displays a strong unfolding intermediate 13% of the time, whereas the truncated domain (yPGKNT) transitions through the intermediate 81% of the time. This effect indicates that the mechanical properties of yPGK cannot be simply deduced from the mechanical properties of its constituents. We also find that Escherichia coli PGK is significantly less mechanically stable as compared to yPGK, contrary to bulk unfolding measurements. Our results support the growing body of observations that the folding behavior of multidomain proteins is difficult to predict based solely on the studies of isolated domains.

Mechanical Unfolding of the High Potential Iron-Sulfur Protein Probed by Single Molecule Atomic Force Microscopy

High potential iron-sulfur protein (HiPIP) is an important category of metalloprotein with a 4Fe-4S cluster center coordinated by four cysteines. Different from other iron-sulfur proteins, the cluster in HiPIP has a high reduction potential, making it an essential electron carrier in bacterial photosynthesis. Here we used single molecule force spectroscopy to investigate the mechanical unfolding of HiPIP and the rupture of the ferric-thiolate bond under different redox potential. We found that the unfolding of HiPIP could be triggered by the rupture of the ferric-thiolate bonds at forces of ∼150 pN, which was lower than those for other iron-sulfur proteins, such as rubredoxin and ferredoxin. Both one-step and two-step unfolding processes were observed in our experiments, indicative of multiple mechanical unfolding pathways for HiPIP. In addition, the oxidation states of the metal center have little effect on the unfolding behavior of HiPiP. Further mutational studies will allow us to elucidate the complete rupture mechanism of the iron-sulfur center and the mechanical unfolding pathways of HiPIP.

Differences in the mechanical unfolding pathways of apo- and copper-bound azurins

Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β–β8 and β7–β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined β4–β7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.

The influence of disulfide bonds on the mechanical stability of proteins is context dependent

Disulfide bonds play a crucial role in proteins, modulating their stability and constraining their conformational dynamics. A particularly important case is that of proteins that need to withstand forces arising from their normal biological function and that are often disulfide bonded. However, the influence of disulfides on the overall mechanical stability of proteins is poorly understood. Here, we used single-molecule force spectroscopy (smFS) to study the role of disulfide bonds in different mechanical proteins in terms of their unfolding forces. For this purpose, we chose the pilus protein FimG from Gram-negative bacteria and a disulfide-bonded variant of the I91 human cardiac titin polyprotein. Our results show that disulfide bonds can alter the mechanical stability of proteins in different ways depending on the properties of the system. Specifically, disulfide-bonded FimG undergoes a 30% increase in its mechanical stability compared with its reduced counterpart, whereas the unfolding force of I91 domains experiences a decrease of 15% relative to the WT form. Using a coarse-grained simulation model, we rationalized that the increase in mechanical stability of FimG is due to a shift in the mechanical unfolding pathway. The simple topology-based explanation suggests a neutral effect in the case of titin. In summary, our results indicate that disulfide bonds in proteins act in a context-dependent manner rather than simply as mechanical lockers, underscoring the importance of considering disulfide bonds both computationally and experimentally when studying the mechanical properties of proteins.

Maltoporin LamB Unfolds β Hairpins along Mechanical Stress-Dependent Unfolding Pathways

Upon mechanical pulling at either terminal end, βbarrel outer membrane proteins stepwise unfold β strands or β hairpins until entirely extracted from the membrane. This unique unfolding pathway hasbeen described for β barrels comprising 8, 14, or 22 β strands. Here we mechanically unfold the 18-stranded β barrel outer membrane protein LamB from Escherichia coli. We find that its mechanical unfolding pathway is shaped by the stepwise unfolding of β hairpins. However, we also observe that βhairpins can unfold groupwise. Thereby, β hairpins unfolding at higher pulling forces show a higher probability to unfold collectively, whereas β hairpins unfolding at lower forces tend to unfold individually. This result suggests that the collective unfolding of β hairpins resembles a far-from-equilibrium process,whereas the unfolding of individual β hairpins describes a closer-to-equilibrium process. Our findings support a direct link between outer membrane protein structure and the unfolding pathway and contribute to a better understanding of their unfolding in response to mechanical stress.

Structural and Mechanistic Insights into the Copper-Modulated Unfolding Pathways of Azurin

Characterization of energy landscape of metalloproteins is challenging because of the subtle changes imparted by metal binding on the stability and conformational flexibility of proteins. Here we present copper-binding induced changes in the mechanical unfolding pathways of azurin, a β-barrel protein with type-I copper center, through a joint experimental-computational study. Single-molecule force spectroscopy experiments reveal an intermediate along the unfolding pathway of apo-azurin in half of population. Steered molecular dynamics simulations attribute the native and intermediate unfolding transition states (TSs) to the rupture of interactions between the pairs of β-strands, 2B-8 and 4-7, respectively. We show that the copper-binding does not change the first TS of azurin and its mechanical properties, but influences the second TS. The rupture of 4-7 β-strand pair is delayed in holo-azurin because of constraints imposed by the copper coordination sphere and occurs after the second TS. Our experimental and computational approach extracts unprecedented details along the unfolding landscape for apo- and holo- forms of azurin.

Determining Factors for the Unfolding Pathway of Peptides, Peptoids, and Peptidic Foldamers

We present a study of the mechanical unfolding pathway of five different oligomers (α-peptide, β-peptide, δ-aromatic-peptides, α/γ-peptides, and β-peptoids), adopting stable helix conformations. Using force-probe molecular dynamics, we identify the determining structural factors for the unfolding pathways and reveal the interplay between the hydrogen bond strength and the backbone rigidity in the stabilization of their helix conformations. On the basis of their behavior, we classify the oligomers in four groups and deduce a set of rules for the prediction of the unfolding pathways of small foldamers.

Force-dependent mechanical unfolding pathways of GFP

We characterize the force-dependent unfolding pathways and intermediate configurations of the green fluorescence protein (GFP) using novel atomistic simulations based on potential energy surface exploration. By using this approach, we are able to unfold GFP to significantly longer end-to-end distances, i.e. 40 nm, as compared to that seen in previous atomistic simulation studies. We find that there are four intermediate states between 5 and 40 nm end-to-end distance, where the intermediate configurations and unfolding pathways are strongly force-dependent. We additionally calculate the force-dependent lifetime of the 14 nm αβ1 intermediate, and demonstrate that it obeys Bell’s formula.

Mechanical unfolding pathway of a model β-peptide foldamer.

Foldamers constructed from oligomers of β-peptides form stable secondary helix structures already for small chain lengths, which makes them ideal candidates for the investigation of the (un)folding of polypeptides. Here, the results of molecular simulations of the mechanical unfolding of a β-heptapeptide in methanol solvent revealing the detailed unfolding pathway are reported. The unfolding process is shown to proceed via a stable intermediate even for such a small system. This result is arrived at performing non-equilibrium force ramp simulations employing different pulling velocities and also using standard calculations of the potential of mean force, i.e., the free energy as a function of the helix elongation. It is thus demonstrated that even with the rather large pulling velocities employed in the force ramp simulations relevant information about the equilibrium kinetics can be obtained. The smallness of the system allows a detailed analysis of the unfolding pathway, which is characterized by an opening of the terminal loops followed by the unfolding of the center. This sequence is in accord with the configurational preferences of the system that also are responsible for the stability of the 314-helix. From an analysis of the distributions of rupture forces and the force spectra, the kinetic rates for both transitions were determined and common models were used to extract geometric quantities describing the free energy landscape of the system.

Exploring the Unfolding Pathway of Maltose Binding Proteins: An Integrated Computational Approach.

Recent single-molecule force spectroscopy experiments on the Maltose Binding Proteins (MBPs) identified four stable structural units, termed unfoldons, that resist mechanical stress and determine the intermediates of the unfolding pathway. In this work, we analyze the topological origin and the dynamical role of the unfoldons using an integrated approach which combines a graph-theoretical analysis of the interaction network of the MBP native-state with steered molecular dynamics simulations. The topological analysis of the native state, while revealing the structural nature of the unfoldons, provides a framework to interpret the MBP mechanical unfolding pathway. Indeed, the experimental pathway can be effectively predicted by means of molecular dynamics simulations with a simple topology-based and low-resolution model of the MBP. The results obtained from the coarse-grained approach are confirmed and further refined by all-atom molecular dynamics.

Single Molecule Force Spectroscopy Reveals the Molecular Mechanical Anisotropy of the FeS4 Metal Center in Rubredoxin

Mechanical anisotropy is an important feature of materials. Depending on the pulling direction, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the pulling direction, a protein can unfold via different mechanical unfolding pathways and exhibit different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecular force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.

Coiled-Coil Probes Capture Mechanical Unfolding Pathways of Individual Proteins

Force-induced unfolding of a consensus ankyrin repeat protein NI6C was suggested to follow a vectorial pathway from the C to the N terminus. We further test this vectorial unfolding hypothesis by introducing, at the gene level, a 70 amino acid coiled-coil (CC) polypeptide probe at different positions along the NI10C sequence and determine the unfolding force extension data of the hybrid proteins with single-molecule atomic force spectroscopy (AFM). The basic idea assumes that the coiled-coil folds independently of the host protein and does not significantly disturb its structure, and that the occurrence and the position of the CC unfolding force peak is indicative of the progress of the unfolding process past the location of the CC probe. First, AFM measurements of the CC probe flanked by I27 domains captured a single ∼35pN unfolding peak characteristic of the unzipping of the coiled-coil. Second, the unfolding force-extension data of the NI8CCI2C hybrid indicated that the unfolding of the CC occurs early in the unfolding process, consistent with the unfolding starting at the C terminus. Third, the force-extension data of the NI4CCI6C indicate that the unfolding of the CC occurs late during the unfolding process, corroborating our vectorial unfolding hypothesis. More generally, our studies suggest that coiled-coil probes maybe very useful to examine mechanical unfolding pathways of many proteins, including large proteins that are rarely studied in bulk folding measurements.

Single Molecule Force Spectroscopy Reveals the Molecular Mechanical Anisotropy of the FeS4 Metal Center in Rubredoxin

Mechanical anisotropy is an important feature of materials. Depending on the direction it is pulled, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the direction along which it is stretched, a protein can unfold via different mechanical unfolding pathways and exhibit vastly different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to the molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in the simplest iron-sulfur protein rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecule atomic force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that the mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small and simple FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.

Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.

A structure-based model fails to probe the mechanical unfolding pathways of the titin I27 domain

We discuss the use of a structure based Cα-Go model and Langevin dynamics to study in detail the mechanical properties and unfolding pathway of the titin I27 domain. We show that a simple Go-model does detect correctly the origin of the mechanical stability of this domain. The unfolding free energy landscape parameters x(u) and ΔG(‡), extracted from dependencies of unfolding forces on pulling speeds, are found to agree reasonably well with experiments. We predict that above v = 10(4) nm/s the additional force-induced intermediate state is populated at an end-to-end extension of about 75 Å. The force-induced switch in the unfolding pathway occurs at the critical pulling speed v(crit) ≈ 10(6)-10(7) nm/s. We argue that this critical pulling speed is an upper limit of the interval where Bell's theory works. However, our results suggest that the Go-model fails to reproduce the experimentally observed mechanical unfolding pathway properly, yielding an incomplete picture of the free energy landscape. Surprisingly, the experimentally observed intermediate state with the A strand detached is not populated in Go-model simulations over a wide range of pulling speeds. The discrepancy between simulation and experiment is clearly seen from the early stage of the unfolding process which shows the limitation of the Go model in reproducing unfolding pathways and deciphering the complete picture of the free energy landscape.