Interplay between Viscoelasticity and Force Rate Affects Sequential Unfolding in Polyproteins Pulled at Constant Velocity.
ABSTRACT: Polyproteins are unique constructs, comprised of folded protein domains in tandem and polymeric linkers. These macromolecules perform under biological stresses by modulating their response through partial unfolding and extending. Although these unfolding events are considered independent, a history dependence of forced unfolding within polyproteins was reported. Here we measure the unfolding of single poly(I91) octamers, complemented with Brownian dynamics simulations, displaying increasing hierarchy in unfolding-foces, accompanied by a decrease in the effective stiffness. This counters the existing understanding that relates stiffness with variations in domain size and probe stiffness, which is expected to reduce the unfolding forces with every consecutive unfolding event. We utilize a simple mechanistic viscoelastic model to show that two effects are combined within a sequential forced unfolding process: the viscoelastic properties of the growing linker chain lead to a hierarchy of the unfolding events, and force-rate application governs the unfolding kinetics.
Project description:We present a novel tensile testing system optimized for the mechanical loading of microliter volume protein hydrogels. Our apparatus incorporates a voice coil servoactuator capable of carrying out fixed velocity extension-relaxation cycles as well as extension step protocols. The setup is equipped with an acrylic cuvette permitting day-long incubations in solution. To demonstrate the functionality of the device, we photochemically crosslinked polyproteins of the I91 immunoglobulin domain from the muscle protein titin to create solid hydrogels that recapitulate elastic properties of muscle. We present data from tensile tests of these low volume biomaterials that support protein unfolding as a main determinant of the elasticity of protein hydrogels. Our results demonstrate the potential use of protein hydrogels as biomaterials whose elastic properties dynamically respond to their environment.
Project description:From a mechanical point of view, a native extracellular matrix (ECM) is viscoelastic. It also possesses time-evolving or dynamic behaviour, since pathophysiological processes such as ageing alter their mechanical properties over time. On the other hand, biomaterial research on mechanobiology has focused mainly on the development of substrates with varying stiffness, with a few recent contributions on time- or space-dependent substrate mechanics. This work reports on a new method for engineering dynamic viscoelastic substrates, i.e., substrates in which viscoelastic parameters can change or evolve with time, providing a tool for investigating cell response to the mechanical microenvironment. In particular, a two-step (chemical and enzymatic) crosslinking strategy was implemented to modulate the viscoelastic properties of gelatin hydrogels. First, gels with different glutaraldehyde concentrations were developed to mimic a wide range of soft tissue viscoelastic behaviours. Then their mechanical behaviour was modulated over time using microbial transglutaminase. Typically, enzymatically induced mechanical alterations occurred within the first 24 h of reaction and then the characteristic time constant decreased although the elastic properties were maintained almost constant for up to seven days. Preliminary cell culture tests showed that cells adhered to the gels, and their viability was similar to that of controls. Thus, the strategy proposed in this work is suitable for studying cell response and adaptation to temporal variations of substrate mechanics during culture.
Project description:Viscoelasticity has emerged as a critical regulator of cell behavior. However, there is an unmet need to develop biomaterials where viscoelasticity can be spatiotemporally controlled to mimic the dynamic and heterogeneous nature of tissue microenvironments. Toward this objective, we developed a modular hyaluronic acid hydrogel combining light-mediated covalent and supramolecular cross-linking to afford spatiotemporal control of network viscoelastic properties. Covalently cross-linked elastic hydrogels or viscoelastic hydrogels combining covalent and supramolecular interactions were fabricated to match healthy and fibrotic liver mechanics. LX-2 human hepatic stellate cells cultured on viscoelastic hydrogels displayed reductions in spreading, actin stress fiber organization, and myocardin-related transcription factor A (MRTF-A) nuclear localization compared to cells on elastic hydrogels. We further demonstrated the dynamic capabilities of our hydrogel system through photo-mediated secondary incorporation of either covalent or supramolecular cross-links to modulate viscoelastic properties. We used photopatterning to create hydrogels with well-controlled patterned regions of stiff elastic mechanics representing fibrotic tissue nodules surrounded by regions of soft viscoelastic hydrogel mimicking healthy tissue. Cells responded to the local mechanics of the patterned substrates with increased spreading in fibrosis-mimicking regions. Together, this work represents an important step forward toward the creation of hydrogel models with spatiotemporal control of both stiffness and viscoelastic cell-instructive cues.
Project description:Materials that can mimic the fibrillar architecture of native extracellular matrix (ECM) while allowing for independent regulation of viscoelastic properties may serve as ideal, artificial ECM (aECM) to regulate cell functions. Here we describe an interpenetrating network of click-functionalized alginate, crosslinked with a combination of ionic and covalent crosslinking, and fibrillar collagen type I. Varying the mode and magnitude of crosslinking enables tunable stiffness and viscoelasticity, while altering neither the hydrogel's microscale architecture nor diffusional transport of molecules with molecular weight relevant to typical nutrients. Further, appropriately timing sequential ionic and covalent crosslinking permits self-assembly of collagen into fibrillar structures within the network. Culture of human mesenchymal stem cells (MSCs) in this mechanically-tunable ECM system revealed that MSC expression of immunomodulatory markers is differentially impacted by the viscoelasticity and stiffness of the matrix. Together, these results describe and validate a novel material system for investigating how viscoelastic mechanical properties of ECM regulate cellular behavior.
Project description:The viscoelastic properties of mammalian cells can vary with biological state, such as during the epithelial-to-mesenchymal (EMT) transition in cancer, and therefore may serve as a useful physical biomarker. To characterize stiffness, conventional techniques use cell contact or invasive probes and as a result are low throughput, labor intensive, and limited by probe placement. Here, we show that measurements of biomass fluctuations in cells using quantitative phase imaging (QPI) provides a probe-free, contact-free method for quantifying changes in cell viscoelasticity. In particular, QPI measurements reveal a characteristic underdamped response of changes in cell biomass distributions versus time. The effective stiffness and viscosity values extracted from these oscillations in cell biomass distributions correlate with effective cell stiffness and viscosity measured by atomic force microscopy (AFM). This result is consistent for multiple cell lines with varying degrees of cytoskeleton disruption and during the EMT. Overall, our study demonstrates that QPI can reproducibly quantify cell viscoelasticity.
Project description:Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.
Project description:<h4>Objective</h4>Viscoelastic properties of articular cartilage have been characterised at physiological frequencies. However, studies investigating the interaction between cartilage and subchondral bone and the influence of underlying bone histomorphometry on the viscoelasticity of cartilage are lacking.<h4>Method</h4>Dynamic Mechanical Analysis (DMA) has been used to quantify the dynamic viscoelasticity of bovine tibial plateau osteochondral cores, over a frequency sweep from 1 to 88 Hz. Specimens (approximately aged between 18 and 30 months) were neither osteoarthritic nor otherwise compromised. A maximum nominal stress of 1.7 MPa was induced. Viscoelastic properties of cores have been compared with that of its components (cartilage and bone) in terms of the elastic and viscous components of both structural stiffness and material modulus. Micro-computed tomography scans were used to quantify the histomorphological properties of the subchondral bone.<h4>Results</h4>Opposing frequency-dependent loss stiffness, and modulus, trends were witnessed for osteochondral tissues: for cartilage it increased logarithmically (P < 0.05); for bone it decreased logarithmically (P < 0.05). The storage stiffness of osteochondral cores was logarithmically frequency-dependent (P < 0.05), however, the loss stiffness was typically frequency-independent (P > 0.05). A linear relationship between the subchondral bone plate (SBP) thickness and cartilage thickness (P < 0.001) was identified. Cartilage loss modulus was linearly correlated to bone mineral density (BMD) (P < 0.05) and bone volume (P < 0.05).<h4>Conclusion</h4>The relationship between the subchondral bone histomorphometry and cartilage viscoelasticity (namely loss modulus) and thickness, have implications for the initiation and progression of osteoarthritis (OA) through an altered ability of cartilage to dissipate energy.
Project description:Viscoelastic characteristics of many materials falling under the category of soft glassy substances, including biological tissue, often exhibit a mechanical complex modulus Y(?) well described by a fractional derivative model: Y(?) = E(i?/?)k, where E = a generalized viscoelastic stiffness; i = (-1)1/2; ? = angular frequency; ? = scaling factor; and k = an exponent valued between 0 and 1. The term "fractional derivative" refers to the value of k: when k = 0 the viscoelastic response is purely elastic, and when k = 1 the response is purely viscous. We provide an analytical derivation of the fractional derivative complex modulus based on the hypothesis that the viscoelastic response arises from many intermittent molecular crosslinks, whose lifetimes longer than a critical threshold lifetime, tcrit, are distributed with an inverse power law proportional to t-(k+2). We demonstrate that E is proportional to the number and stiffness of crosslinks formed at any moment; the scaling factor ? is equivalent to reciprocal of tcrit; and the relative mean lifetime of the attached crosslinks is inversely proportional to the parameter k. To test whether electrostatic molecular bonds could be responsible for the fractional derivative viscoelasticity, we used chemically skinned human skeletal muscle as a one-dimensional model of a soft glassy substance. A reduction in ionic strength from 175 to 110 mEq resulted in a larger E with no change in k, consistent with a higher probability of interfilament molecular interactions. Thick to thin filament spacing was reduced by applying 4% w/v of the osmolyte Dextran T500, which also resulted in a larger E, indicating a greater probability of crosslink formation in proportion to proximity. A 10°C increase in temperature resulted in an increase in k, which corresponded to a decrease in cross-bridge attachment lifetime expected with higher temperatures. These theoretical and experimental results suggest that the fractional derivative viscoelasticity observed in some biological tissue arises as a mechanical consequence of electrostatic interactions, whose longest lifetimes are distributed with an inverse power law.
Project description:Cerebral palsy (CP) is a neurodevelopmental disorder that results in functional motor impairment and disability in children. CP is characterized by neural injury though many children do not exhibit brain lesions or damage. Advanced structural MRI measures may be more sensitively related to clinical outcomes in this population. Magnetic resonance elastography (MRE) measures the viscoelastic mechanical properties of brain tissue, which vary extensively between normal and disease states, and we hypothesized that the viscoelasticity of brain tissue is reduced in children with CP. Using a global region-of-interest-based analysis, we found that the stiffness of the cerebral gray matter in children with CP is significantly lower than in typically developing (TD) children, while the damping ratio of gray matter is significantly higher in CP. A voxel-wise analysis confirmed this finding, and additionally found stiffness and damping ratio differences between groups in regions of white matter. These results indicate that there is a difference in brain tissue health in children with CP that is quantifiable through stiffness and damping ratio measured with MRE. Understanding brain tissue mechanics in the pediatric CP population may aid in the diagnosis and evaluation of CP.
Project description:Transient elastography quantifies the propagation of a mechanically generated shear wave within a soft tissue, which can be used to characterize the elasticity and viscosity parameters of the tissue. The aim of our study was to combine numerical simulation and clinical assessment to define a viscoelastic index of liver tissue to improve the quality of early diagnosis of liver fibrosis. This is clinically relevant, as early fibrosis is reversible. We developed an idealized two-dimensional axisymmetric finite element model of the liver to evaluate the effects of different viscoelastic values on the propagation characteristics of the shear wave. The diagnostic value of the identified viscoelastic index was verified against the clinical data of 99 patients who had undergone biopsy and routine blood tests for staging of liver disease resulting from chronic hepatitis B infection. Liver stiffness measurement (LSM) and the shear wave attenuation fitting coefficient (AFC) were calculated from the ultrasound data obtained by performing transient elastography. Receiver operating curve analysis was used to evaluate the reliability and diagnostic accuracy of LSM and AFC. Compared to LSM, the AFC provided a higher diagnostic accuracy to differentiate early stages of liver fibrosis, namely F1 and F2 stages, with an overall specificity of 81.48%, sensitivity of 83.33% and diagnostic accuracy of 81.82%. AFC was influenced by the level of LSM, ALT. However, there are no correlation between AFC and Age, BMI, TBIL or DBIL. Quantification of the viscoelasticity of liver tissue provides reliable measurement to identify and differentiate early stages of liver fibrosis.