Mechanical activation of a multimeric adhesive protein through domain conformational change.

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

Direct observation of multiple pathways of single-stranded DNA stretching.

We observed multiple pathways of stretching single-stranded polydeoxynucleotides, poly(dA). Poly(dA) has been shown to undergo unique transitions under mechanical force, and such transitions were attributed to the stacking characteristics of poly(dA). Using single-molecule manipulation studies, we found that poly(dA) has two stretching pathways at high forces. The previously observed pathway has a free energy that is less than what is expected of single-stranded DNA with a random sequence, indicating the existence of a novel conformation of poly(dA) at large extensions. We also observed stepwise transitions between the two pathways by pulling the molecule with constant force, and found that the transitions are cooperative. These results suggest that the unique mechanical property of poly(dA) may play an important role in biological processes such as gene expression.

Dependence of Membrane Tether Strength on Substrate Rigidity Probed by Single-Cell Force Spectroscopy.

Substrate rigidity modulates cell mechanics, which affect cell migration and proliferation. Quantifying the effects of substrate rigidity on cancer cell mechanics requires a quantifiable parameter that can be measured for individual cells, as well as a substrate platform with rigidity being the only variable. Here we used single-cell force spectroscopy to pull cancer cells on substrates varying only in rigidity, and extracted a parameter from the force-distance curves to be used to quantify the properties of membrane tethers. Our results showed that tether force increases with substrate rigidity until it reaches its asymptotic limit. The variations are similar for all three cancer cell lines studied, and the largest change occurs in the rigidity regions of softer tissues, indicating a universal response of cancer cell elasticity to substrate rigidity.

Mechanical Responses of Breast Cancer Cells to Substrates of Varying Stiffness Revealed by Single-Cell Measurements.

How cancer cells respond to different mechanical environments remains elusive. Here, we investigated the tension in single focal adhesions of MDA-MB-231 (metastatic breast cancer cells) and MCF-10A (normal human breast cells) cells on substrates of varying stiffness using single-cell measurements. Tension measurements in single focal adhesions using an improved FRET-based tension sensor showed that the tension in focal adhesions of MDA-MB-231 cells increased on stiffer substrates while the tension in MCF-10A cells exhibited no apparent change against the substrate stiffness. Viscoelasticity measurements using magnetic tweezers showed that the power-law exponent of MDA-MB-231 cells decreased on stiffer substrates whereas MCF-10A cells had similar exponents throughout the whole stiffness, indicating that MDA-MB-231 cells change their viscoelasticity on stiffer substrates. Such changes in tension in focal adhesions and viscoelasticity against the substrate stiffness represent an adaptability of cancer cells in mechanical environments, which can facilitate the metastasis of cancer cells to different tissues.

Analyzing single-molecule manipulation experiments.

Single-molecule manipulation studies can provide quantitative information about the physical properties of complex biological molecules without ensemble artifacts obscuring the measurements. We demonstrate computational techniques which aim at more fully utilizing the wealth of information contained in noisy experimental time series. The "noise" comes from multiple sources e.g., inherent thermal motion, instrument measurement error, etc. The primary focus of this paper is a methodology that uses time domain based methods to extract the effective molecular friction from single-molecule pulling data. We studied molecules composed of eight tandem repeat titin I27 domains, but the modeling approaches have applicability to other single-molecule mechanical studies. The merits and challenges associated with applying such a computational approach to existing single-molecule manipulation data are also discussed.

Quantifying multiscale noise sources in single-molecule time series.

When analyzing single-molecule data, a low-dimensional set of system observables typically serves as the observational data. We calibrate stochastic dynamical models from time series that record such observables. Numerical techniques for quantifying noise from multiple time scales in a single trajectory, including experimental instrument and inherent thermal noise, are demonstrated. The techniques are applied to study time series coming from both simulations and experiments associated with the nonequilibrium mechanical unfolding of titin's I27 domain. The estimated models can be used for several purposes, (1) detect dynamical signatures of "rare events" by analyzing the effective diffusion and force as a function of the monitored observable, (2) quantify the influence that conformational degrees of freedom, which are typically difficult to directly monitor experimentally, have on the dynamics of the monitored observable, (3) quantitatively compare the inherent thermal noise to other noise sources, for example, instrument noise, variation induced by conformational heterogeneity, and so forth, (4) simulate random quantities associated with repeated experiments, and (5) apply pathwise, that is, trajectory-wise, hypothesis tests to assess the goodness-of-fit of the models and even detect conformational transitions in noisy signals. These items are all illustrated with several examples.

Temperature and chemical denaturant dependence of forced unfolding of titin I27.

Single-molecule force measurement opens a new door for investigating detailed biomolecular interactions and their thermodynamic properties by pulling molecules apart while monitoring the force exerted on them. Recent advances in the nonequilibrium work theorem allows one to determine the free-energy landscapes of these events. Such information is valuable for understanding processes such as protein and RNA folding and receptor-ligand binding. Here, we used force as a physical parameter under the traditional chemical and temperature denaturing environment to alter the protein folding energy landscape and compared the change in the unfolding free-energy barrier of the I27 domain of human cardiac titin. We found that the trends in protein unfolding free-energy barriers are consistent for single-molecule force measurements and bulk chemical and temperature studies. The results suggest that the information from single-molecule pulling experiments are meaningful and useful for understanding the mechanism of folding of titin I27.

Velocity convergence of free energy surfaces from single-molecule measurements using Jarzynski's equality.

We studied the velocity dependence of mechanical unfolding of single protein molecules with the atomic force microscope. We showed that with enough realizations, the free energy surfaces reconstructed from Jarzynski's equality converge with respect to pulling velocity, in good agreement with theory. Using the I27 domain of titin as an example, we estimated the required number of realizations for a given pulling velocity, and suggested the optimal range of velocities for single-molecule experiments. The results demonstrate that Jarzynski's equality is a powerful and practical tool for reconstructing free energy landscapes.

Quantifying DNA melting transitions using single-molecule force spectroscopy.

We stretched a DNA molecule using atomic force microscope and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA (ssDNA). We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

Correlating Conformational Dynamics with the Von Willebrand Factor Reductase Activity of Factor H Using Single Molecule Force Measurements.

Activation of proteins often involves conformational transitions, and these switches are often difficult to characterize in multidomain proteins. Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kD), is a complement control protein that regulates the activity of the alternative complement pathway. Different preparations of FH can also reduce the disulfide bonds linking large Von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In contrast, commercially available purified FH (pFH) has little or no VWF reductase activity unless the pFH is chemically modified by either ethylenediaminetetraacetic acid (EDTA) or urea. We used atomic force microscopy single molecule force measurements to investigate different forms of FH, including recombinant FH and pFH, in the presence or absence of EDTA and urea, and to correlate the conformational changes to its activities. We found that the FH conformation depends on the method used for sample preparation, which affects the VWF reductase activity of FH.

Defects can increase the melting temperature of DNA-nanoparticle assemblies.

DNA-gold nanoparticle assemblies have shown promise as an alternative technology to DNA microarrays for DNA detection and RNA profiling. Understanding the effect of DNA sequences on the melting temperature of the system is central to developing reliable detection technology. We studied the effects of DNA base-pairing defects, such as mismatches and deletions, on the melting temperature of DNA-nanoparticle assemblies. We found that, contrary to the general assumption that defects lower the melting temperature of DNA, some defects increase the melting temperature of DNA-linked nanoparticle assemblies. The effects of mismatches and deletions were found to depend on the specific base pair, the sequence, and the location of the defects. Our results demonstrate that the surface-bound DNA exhibit hybridization behavior different from that of free DNA. Such findings indicate that a detailed understanding of DNA-nanoparticle assembly phase behavior is required for quantitative interpretation of DNA-nanoparticle aggregation.

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix.

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20 nm and a diameter of 2-4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young's modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor.

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100dynes/cm^{2} for 3 min at 37^{∘}C). We conclude that under the shear conditions used (100dynes/cm^{2} for 3 min at 37^{∘}C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

Detecting the Biopolymer Behavior of Graphene Nanoribbons in Aqueous Solution.

Graphene nanoribbons (GNR), can be prepared in bulk quantities for large-area applications by reducing the product from the lengthwise oxidative unzipping of multiwalled carbon nanotubes (MWNT). Recently, the biomaterials application of GNR has been explored, for example, in the pore to be used for DNA sequencing. Therefore, understanding the polymer behavior of GNR in solution is essential in predicting GNR interaction with biomaterials. Here, we report experimental studies of the solution-based mechanical properties of GNR and their parent products, graphene oxide nanoribbons (GONR). We used atomic force microscopy (AFM) to study their mechanical properties in solution and showed that GNR and GONR have similar force-extension behavior as in biopolymers such as proteins and DNA. The rigidity increases with reducing chemical functionalities. The similarities in rigidity and tunability between nanoribbons and biomolecules might enable the design and fabrication of GNR-biomimetic interfaces.

DNA under Force: Mechanics, Electrostatics, and Hydration.

Quantifying the basic intra- and inter-molecular forces of DNA has helped us to better understand and further predict the behavior of DNA. Single molecule technique elucidates the mechanics of DNA under applied external forces, sometimes under extreme forces. On the other hand, ensemble studies of DNA molecular force allow us to extend our understanding of DNA molecules under other forces such as electrostatic and hydration forces. Using a variety of techniques, we can have a comprehensive understanding of DNA molecular forces, which is crucial in unraveling the complex DNA functions in living cells as well as in designing a system that utilizes the unique properties of DNA in nanotechnology.

Reconstructing multiple free energy pathways of DNA stretching from single molecule experiments.

Free energy landscapes provide information on the dynamics of proteins and nucleic acid folding. It has been demonstrated that such landscapes can be reconstructed from single molecule force measurement data using Jarzynski's equality, which requires only stretching data. However, when the process is reversible, the Crooks fluctuation theorem combines both stretch and relaxation force data for the analysis and can offer more rapid convergence of free energy estimates of different states. Here we demonstrate that, similar to Jarzynski's equality, the Crooks fluctuation theorem can be used to reconstruct the full free energy landscapes. In addition, when the free energy landscapes exhibit multiple folding pathways, one can use Jarzynski's equality to reconstruct individual free energy pathways if the experimental data show distinct work distributions. We applied the method to reconstruct the overstretching transition of poly(dA) to demonstrate that the nonequilibrium work theorem combined with single molecule force measurements provides a clear picture of the free energy landscapes.

Multiscale mechanobiology: mechanics at the molecular, cellular, and tissue levels.

Mechanical force is present in all aspects of living systems. It affects the conformation of molecules, the shape of cells, and the morphology of tissues. All of these are crucial in architecture-dependent biological functions. Nanoscience of advanced materials has provided knowledge and techniques that can be used to understand how mechanical force is involved in biological systems, as well as to open new avenues to tailor-made bio-mimetic materials with desirable properties.In this article, we describe models and show examples of how force is involved in molecular functioning, cell shape patterning, and tissue morphology.

Understanding the physics of DNA using nanoscale single-molecule manipulation.

Processes for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code.

Phase Transition and Optical Properties of DNA-Gold Nanoparticle Assemblies.

We review recent work on DNA-linked gold nanoparticle assemblies. The synthesis, properties, and phase behavior of such DNA-gold nanoparticle assemblies are described. These nanoparticle assemblies have strong optical extinction in the ultraviolet and visible light regions; hence, the technique is used to study the kinetics and phase transitions of DNA-gold nanoparticle assemblies. The melting transition of DNA-gold nanoparticle assemblies shows unusual trends compared to those of free DNA. The phase transitions are influenced by many parameters, such as nanoparticle size, DNA sequence, DNA grafting density, DNA linker length, interparticle distance, base pairing defects, and disorders. The physics of the DNA-gold nanoparticle assemblies can be understood in terms of the phase behavior of complex fluids, with the colloidal gold interaction potential dominated by DNA hybridization energies.

Experimental free energy surface reconstruction from single-molecule force spectroscopy using Jarzynski's equality.

We used the atomic force microscope to manipulate and unfold individual molecules of the titin I27 domain and reconstructed its free energy surface using Jarzynski's equality. The free energy surface for both stretching and unfolding was reconstructed using an exact formula that relates the nonequilibrium work fluctuations to the molecular free energy. In addition, the unfolding free energy barrier, i.e., the activation energy, was directly obtained from experimental data for the first time. This Letter demonstrates that Jarzynski's equality can be used to analyze nonequilibrium single-molecule experiments, and to obtain the free energy surfaces for molecular systems, including interactions for which only nonequilibrium work can be measured.