Single-molecule Force Spectroscopy Studies Research Articles

Facile Method of Constructing Polyproteins for Single-Molecule Force Spectroscopy Studies

Constructing polyproteins consisting of identical tandem repeats of proteins provides an unambiguous method of investigating the mechanical properties of proteins at the single-molecule level using force spectroscopy techniques. Here we report a maleimide-thiol coupling-based facile method of constructing polyproteins for single-molecule force spectroscopy studies on the mechanical properties of proteins. This method allows for the construction of polyproteins in an efficient fashion under room temperature. The resultant thioether bonds are resistant to reduction and make it possible to carry out single-molecule force spectroscopy studies under various redox conditions. This novel method complements existing polyprotein engineering methods and can be easily applied to a wide variety of proteins.

Propagation of Load Through Proteins via Steered Molecular Dynamics: Effect on Fluctuation Dynamics and Architecture

Mechanical load in the form of externally applied forces and/or moments are known to regulate biochemical activity in proteins through induced conformational changes exposing cryptic binding sites, altered kinetics, and, in extreme cases, unfolding. It is unknown, however, how load propagates through protein structure from remotely applied forces to local regions: do loads manifest as changes in fluctuations, architecture or both? Single molecule force spectroscopy (SMFS) studies have demonstrated protein unfolding with nanometer- and piconewton-level resolution, yet provide little information about the intra-protein pathways of load. SMFS studies of Green Flourescent Protein (GFP) have demonstrated the anisotropy of deformation, providing an ideal test system for studying the structural response of proteins to load. To explore the manifestation of load in proteins via simulation of a controlled experiment, constant-force steered molecular dynamics (SMD) are used to generate probability density functions (PDFs) at atomic-level resolution describing the structure of GFP when subjected to simple, sub-unfolding, externally applied loads along the vectors used in SMFS studies. Here the focus is on describing protein structure (both vibrational and architectural) with increasing forces; the experimental unfolding values provide a top-force validation of the simulation results. Total data include PDFs for both equilibrium and non-equilibrium loaded scenarios permitting comparison for the effect of loads. Data are analyzed for principal components (via PCA) to determine fluctuation dynamics and changes in structure architecture along the spectrum of applied loads. PCA data allow comparison of both fluctuation amplitude and direction through the power spectra of mode covariance overlap. Results indicate areas of increased strain under load, predicting locations of unfolding via increased strain and demonstrating the anisotropic pathways of load at sub-unfolding loads. Intra-molecular constitutive properties are calculated as derivatives of free energy.

Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 microm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds >or=100 microm/s with minimal viscous drag effects. We have used FLUENT, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates >or=10(6) pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.

Precision Force Spectroscopy of Bacteriorhodopsin

Single-molecule force spectroscopy studies have produced rich insights into the unfolding of individual proteins and nucleic acid structures. In a typical force spectroscopy experiment, an AFM tip is coupled to a surface-adsorbed protein by pressing the tip into it. Force-extension curves are then generated by retracting the tip at a constant velocity using a piezoelectric (PZT) stage. Force is measured by cantilever deflection. Extension, or more precisely tip-sample separation, is deduced from the PZT stage position used to control the vertical tip position. Thus, this deduced extension is sensitive to the vertical mechanical drift of the AFM assembly (∼10 nm/min). We have previously developed an ultrastable AFM in which the tip and the sample positions are independently measured by, and stabilized with respect to, a pair of laser foci in three dimensions. These lasers establish a local reference frame that is insensitive to long-term mechanical drift of the AFM assembly. This new measurement of position is complementary to the cantilever deflection sensing, which measures force. We have now extended the ultrastable AFM capabilities into liquid and can routinely mechanically unfold proteins at slow pulling velocities (2 nm/s), which allows averaging to increase precision. We can also stop pulling altogether and hold the molecule at constant force while independently measuring tip-sample separation (σ = 0.2 nm, Δf = 1-50 Hz). Alternatively, we can stabilize tip-sample separation and measure force (σ = 5 pN, Δf = 1-50 Hz) over 100s of seconds . Using these techniques, we are studying the unfolding and re-folding of bacteriorhodopsin (BR), a model transmembrane protein.

Single‐Molecule Mechanical Unfolding of Amyloidogenic β2‐Microglobulin: The Force‐Spectroscopy Approach

The recombinant production of a novel chimeric polyprotein is described. The new protein contains either wild-type beta(2)-microglobulin (beta(2)m) or its truncated variant (DeltaN6 beta(2)m) (see picture). Structural characterization is achieved by means of single-molecule force spectroscopy studies of specific beta(2)m regions which could be involved in amyloidogenesis.

Protonated Nanoparticle Surface Governing Ligand Tethering and Cellular Targeting

Nanoparticles have shown tremendous potential for effective drug delivery due to their tiny size and cell membrane penetration capabilities. Cellular targeting with nanoparticles is often achieved by surface modifications followed by ligand conjugation. However, the efficiency of the nanoparticles reaching the target cells and getting internalized depends on the stability of targeting ligands and the chemical nature of the ligand nanoparticle binding. Recent advancements in nanobiomaterials research have proven the superoxide dismutase (SOD) mimetic activity of cerium oxide nanoparticles (CNPs) in protecting cells against oxidative stress. Due to their excellent biocompatibility, CNPs can be used as a potential drug carrier that can transport and release drugs to the malignant sites. Here we combine single molecule force spectroscopy (SMFS) and density functional theory (DFT) simulations to understand the interaction between transferrin, a ligand protein overexpressed in cancer cells, and CNPs. SMFS studies demonstrate an increase in the transferrin adhesion to the nanoparticles' surface with an increase in positive zeta potential of CNPs. Binding energy values obtained from DFT calculations predict an increase in bond strength between the transferrin and CNPs upon surface protonation and charge modification. Transferrin-conjugated CNPs were tested for their binding stability and preferential cellular uptake efficiency by incubating them with human lung cancer cells (A549) and normal embryo lung cells (WI-38). The results demonstrate the importance of tuning the surface properties of nanoparticles for better ligand adsorption and cellular uptake.

Recognition Imaging and Highly Ordered Molecular Templating of Bacterial S-Layer Nanoarrays Containing Affinity-Tags

Functional nanoarrays were fabricated using the chimeric bacterial cell surface layer (S-layer) protein rSbpA fused with the affinity tag Strep-tagII and characterized using various atomic force microscopy (AFM) techniques in aqueous environment. The accessibility of Strep-tagII was verified by single-molecule force spectroscopy studies employing Strep-Tactin as specific ligand. Simultaneous topography and recognition imaging (TREC) of the nanoarray yielded high resolution maps of the Strep-tagll binding sites with a positional accuracy of 1.5 nm. The nanoarrays were used as template for constructing highly ordered molecular binding blocks.

Stabilization Provided by Neighboring Strands Is Critical for the Mechanical Stability of Proteins

Single-molecule force spectroscopy studies and steered molecular dynamics simulations have revealed that protein topology and pulling geometry play important roles in determining the mechanical stability of proteins. Most studies have focused on local interactions that are associated with the force-bearing β-strands. Interactions mediated by neighboring strands are often overlooked. Here we use Top7 and barstar as model systems to illustrate the critical importance of the stabilization effect provided by neighboring β-strands on the mechanical stability. Using single-molecule atomic force microscopy, we showed that Top7 and barstar, which have similar topology in their force-bearing region, exhibit vastly different mechanical-stability characteristics. Top7 is mechanically stable and unfolds at ∼150pN, whereas barstar is mechanically labile and unfolds largely below 50pN. Steered molecular dynamics simulations revealed that stretching force peels one force-bearing strand away from barstar to trigger unfolding, whereas Top7 unfolds via a substructure-sliding mechanism. This previously overlooked stabilization effect from neighboring β-strands is likely to be a general mechanism in protein mechanics and can serve as a guideline for the de novo design of proteins with significant mechanical stability and novel protein topology.

AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptideglucagon

We report on the mechanical characterization of individual mature amyloid fibrils byatomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy(SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptidehormone glucagon, were found to display a reversible elastic behaviour. Based onAFM morphology and SMFS studies, we suggest that the observed elasticity isdue to a force-induced conformational transition which is reversible due to theβ-helical conformation of protofibrils, allowing a high degree of extension. The elasticproperties of such mature fibrils contribute to their high stability, suggesting that theinternal hydrophobic interactions of amyloid fibrils are likely to be of fundamentalimportance in the assembly of amyloid fibrils and therefore for the understanding of theprogression of their associated pathogenic disorders. In addition, such biological amyloidfibril structures with highly stable mechanical properties can potentially be used toproduce nanofibres (nanowires) that may be suitable for nanotechnological applications.

Immunochemical and single molecule force spectroscopy studies of specific interaction between the laminin binding protein and the West Nile virus surface glycoprotein E domain II

ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.

An RNA toolbox for single-molecule force spectroscopy studies.

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.

Proteoglycan Mechanics Studied by Single-molecule Force Spectroscopy of Allotypic Cell Adhesion Glycans

Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.

Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.