CellDynaMo-stochastic reaction-diffusion-dynamics model: Application to search-and-capture process of mitotic spindle assembly.

We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly.

Regulatory element in fibrin triggers tension-activated transition from catch to slip bonds.

Fibrin formation and mechanical stability are essential in thrombosis and hemostasis. To reveal how mechanical load impacts fibrin, we carried out optical trap-based single-molecule forced unbinding experiments. The strength of noncovalent A:a knob-hole bond stabilizing fibrin polymers first increases with tensile force (catch bonds) and then decreases with force when the force exceeds a critical value (slip bonds). To provide the structural basis of catch-slip-bond behavior, we analyzed crystal structures and performed molecular modeling of A:a knob-hole complex. The movable flap (residues γ295 to γ305) containing the weak calcium-binding site γ2 serves as a tension sensor. Flap dissociation from the B domain in the γ-nodule and translocation to knob 'A' triggers hole 'a' closure, resulting in the increase of binding affinity and prolonged bond lifetimes. The discovery of biphasic kinetics of knob-hole bond rupture is quantitatively explained by using a theory, formulated in terms of structural transitions in the binding pocket between the low-affinity (slip) and high-affinity (catch) states. We provide a general framework to understand the mechanical response of protein pairs capable of tension-induced remodeling of their association interface. Strengthening of the A:a knob-hole bonds at 30- to 40-pN forces might favor formation of nascent fibrin clots subject to hydrodynamic shear in vivo.

Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling.

Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots, yet the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing of fibrin fibers. Here the packing structure of fibrin fibers is quantitatively assessed by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin reconstituted under a wide range of conditions with computational molecular modeling of fibrin protofibrils. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbß3.

Binding of soluble fibrinogen to the activated conformation of the integrin αIIbß3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbß3 and make substantial contributions to αIIbß3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbß3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbß3 conformers at the interface between the αIIb ß-propeller domain and the ß3 ßI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbß3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbß3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbß3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the αIIbß3-peptide complexes revealed that the overall stability of the αIIbß3-cRGDFK complex was comparable with that of the αIIbß3-γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbß3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbß3 with immobilized fibrinogen and fibrin.

Dynamic Transition from α-Helices to ß-Sheets in Polypeptide Coiled-Coil Motifs.

We carried out dynamic force manipulations in silico on a variety of coiled-coil protein fragments from myosin, chemotaxis receptor, vimentin, fibrin, and phenylalanine zippers that vary in size and topology of their α-helical packing. When stretched along the superhelical axis, all superhelices show elastic, plastic, and inelastic elongation regimes and undergo a dynamic transition from the α-helices to the ß-sheets, which marks the onset of plastic deformation. Using the Abeyaratne-Knowles formulation of phase transitions, we developed a new theoretical methodology to model mechanical and kinetic properties of protein coiled-coils under mechanical nonequilibrium conditions and to map out their energy landscapes. The theory was successfully validated by comparing the simulated and theoretical force-strain spectra. We derived the scaling laws for the elastic force and the force for α-to-ß transition, which can be used to understand natural proteins' properties as well as to rationally design novel biomaterials of required mechanical strength with desired balance between stiffness and plasticity.

Fluctuating Nonlinear Spring Model of Mechanical Deformation of Biological Particles.

The mechanical properties of virus capsids correlate with local conformational dynamics in the capsid structure. They also reflect the required stability needed to withstand high internal pressures generated upon genome loading and contribute to the success of important events in viral infectivity, such as capsid maturation, genome uncoating and receptor binding. The mechanical properties of biological nanoparticles are often determined from monitoring their dynamic deformations in Atomic Force Microscopy nanoindentation experiments; but a comprehensive theory describing the full range of observed deformation behaviors has not previously been described. We present a new theory for modeling dynamic deformations of biological nanoparticles, which considers the non-linear Hertzian deformation, resulting from an indenter-particle physical contact, and the bending of curved elements (beams) modeling the particle structure. The beams' deformation beyond the critical point triggers a dynamic transition of the particle to the collapsed state. This extreme event is accompanied by a catastrophic force drop as observed in the experimental or simulated force (F)-deformation (X) spectra. The theory interprets fine features of the spectra, including the nonlinear components of the FX-curves, in terms of the Young's moduli for Hertzian and bending deformations, and the structural damage dependent beams' survival probability, in terms of the maximum strength and the cooperativity parameter. The theory is exemplified by successfully describing the deformation dynamics of natural nanoparticles through comparing theoretical curves with experimental force-deformation spectra for several virus particles. This approach provides a comprehensive description of the dynamic structural transitions in biological and artificial nanoparticles, which is essential for their optimal use in nanotechnology and nanomedicine applications.

SOP-GPU: influence of solvent-induced hydrodynamic interactions on dynamic structural transitions in protein assemblies.

Hydrodynamic interactions (HI) are incorporated into Langevin dynamics of the Cα -based protein model using the Truncated Expansion approximation (TEA) to the Rotne-Prager-Yamakawa diffusion tensor. Computational performance of the obtained GPU realization demonstrates the model's capability for describing protein systems of varying complexity (10(2) -10(5) residues), including biological particles (filaments, virus shells). Comparison of numerical accuracy of the TEA versus exact description of HI reveals similar results for the kinetics and thermodynamics of protein unfolding. The HI speed up and couple biomolecular transitions through cross-communication among protein domains, which result in more collective displacements of structure elements governed by more deterministic (less variable) dynamics. The force-extension/deformation spectra from nanomanipulations in silico exhibit sharper force signals that match well the experimental profiles. Hence, biomolecular simulations without HI overestimate the role of tension/stress fluctuations. Our findings establish the importance of incorporating implicit water-mediated many-body effects into theoretical modeling of dynamic processes involving biomolecules. © 2016 Wiley Periodicals, Inc.

Assembly and Mechanical Properties of the Cargo-Free and Cargo-Loaded Bacterial Nanocompartment Encapsulin.

Prokaryotes mostly lack membranous compartments that are typical of eukaryotic cells, but instead, they have various protein-based organelles. These include bacterial microcompartments like the carboxysome and the virus-like nanocompartment encapsulin. Encapsulins have an adaptable mechanism for enzyme packaging, which makes it an attractive platform to carry a foreign protein cargo. Here we investigate the assembly pathways and mechanical properties of the cargo-free and cargo-loaded nanocompartments, using a combination of native mass spectrometry, atomic force microscopy and multiscale computational molecular modeling. We show that encapsulin dimers assemble into rigid single-enzyme bacterial containers. Moreover, we demonstrate that cargo encapsulation has a mechanical impact on the shell. The structural similarity of encapsulins to virus capsids is reflected in their mechanical properties. With these robust mechanical properties encapsulins provide a suitable platform for the development of nanotechnological applications.

Molecular mechanisms, thermodynamics, and dissociation kinetics of knob-hole interactions in fibrin.

Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs 'A' and 'B' in the central nodule of fibrin monomer to complementary holes 'a' and 'b' in the γ- and ß-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using molecular dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob 'A' mimetic) and GHRP (knob 'B' mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; however, the dissociation kinetics were sensitive to variations in acidity (pH 5-7) and temperature (T = 25-37 °C). There were similar structural changes in holes 'a' and 'b' during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of the interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an "all-or-none" transition as it occurred through distinct two-step or single step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH 7 than at pH 5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH 7 but stronger, tighter, and less compliant at pH 5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T = 37 °C) compared with T = 25 °C due to the helix-to-coil transition in loop I that helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.

Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4.

Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (k(off) = 0.0031 and 0.0029 s(-1)). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4(K50E), which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.

Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico.

Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral noncovalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physicochemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversible dissociation of lateral bonds followed by irreversible dissociation of the longitudinal bonds. We have determined the free energies of dissociation of the lateral (6.9 ± 0.4 kcal/mol) and longitudinal (14.9 ± 1.5 kcal/mol) tubulin-tubulin bonds. These values in conjunction with the large flexural rigidity of tubulin protofilaments obtained (18,000-26,000 pN·nm(2)) support the idea that the disassembling microtubule is capable of generating a large mechanical force to move chromosomes during cell division. Our computational modeling offers a comprehensive quantitative platform to link molecular tubulin characteristics with the physiological behavior of microtubules. The developed in silico nanoindentation method provides a powerful tool for the exploration of biomechanical properties of other cytoskeletal and multiprotein assemblies.

Structural transitions and energy landscape for Cowpea Chlorotic Mottle Virus capsid mechanics from nanomanipulation in vitro and in silico.

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔHind = 11.5-12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔSind = 5.1-5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

Resolving two-dimensional kinetics of the integrin αIIbß3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbß3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbß3 molecules and forced dissociation of an αIIbß3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbß3 pre-exists in at least two states that differ in their zero force on-rates (k(on1) = 1.4 × 10(-4) and k(on2) = 2.3 × 10(-4) µm(2)/s), off-rates (k(off1) = 2.42 and k(off2) = 0.60 s(-1)), and dissociation constants (K(d)(1) = 1.7 × 10(4) and K(d)(2) = 2.6 × 10(3) µm(-2)). The integrin activator Mn(2+) changed the on-rates and affinities (K(d)(1) = 5 × 10(4) and K(d)(2) = 0.3 × 10(3) µm(-2)) but did not affect the off-rates. The strength of αIIbß3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbß3-fibrinogen complex characterized by a faster on-rate. Upon Mn(2+)-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbß3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbß3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.

Interrelated effects of chromosome size, mechanics, number, location-orientation and polar ejection force on the spindle accuracy: a 3D computational study.

The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to address two questions: how rapidly the microtubule-kinetochore connections are made, and how accurate these connections are. In most previous numerical simulations, the model geometry was drastically simplified. Using the CellDynaMo computational platform, we previously introduced a geometrically and mechanically realistic 3D model of the prometaphase mitotic spindle, and used it to evaluate thermal noise and microtubule kinetics effects on the capture of a single chromosome. Here, we systematically investigate how geometry and mechanics affect a spindle assembly's speed and accuracy, including nuanced distinctions between merotelic, mero-amphitelic, and mero-syntelic chromosomes. We find that softening of the centromere spring improves accuracy for short chromosome arms, but accuracy disappears for long chromosome arms. Initial proximity of chromosomes to one spindle pole makes assembly accuracy worse, while initial chromosome orientation matters less. Chromokinesins, added onto flexible chromosome arms, allow modeling of the polar ejection force, improving a spindle assembly's accuracy for a single chromosome. However, spindle space crowding by multiple chromosomes worsens assembly accuracy. Our simulations suggest that the complex microtubule network of the early spindle is key to rapid and accurate assembly.

Mechanical fatigue testing in silico: Dynamic evolution of material properties of nanoscale biological particles.

Biological particles have evolved to possess mechanical characteristics necessary to carry out their functions. We developed a computational approach to "fatigue testing in silico", in which constant-amplitude cyclic loading is applied to a particle to explore its mechanobiology. We used this approach to describe dynamic evolution of nanomaterial properties and low-cycle fatigue in the thin spherical encapsulin shell, thick spherical Cowpea Chlorotic Mottle Virus (CCMV) capsid, and thick cylindrical microtubule (MT) fragment over 20 cycles of deformation. Changing structures and force-deformation curves enabled us to describe their damage-dependent biomechanics (strength, deformability, stiffness), thermodynamics (released and dissipated energies, enthalpy, and entropy) and material properties (toughness). Thick CCMV and MT particles experience material fatigue due to slow recovery and damage accumulation over 3-5 loading cycles; thin encapsulin shells show little fatigue due to rapid remodeling and limited damage. The results obtained challenge the existing paradigm: damage in biological particles is partially reversible owing to particle's partial recovery; fatigue crack may or may not grow with each loading cycle and may heal; and particles adapt to deformation amplitude and frequency to minimize the energy dissipated. Using crack size to quantitate damage is problematic as several cracks might form simultaneously in a particle. Dynamic evolution of strength, deformability, and stiffness, can be predicted by analyzing the cycle number (N) dependent damage, [Formula: see text] , where α is a power law and Nf is fatigue life. Fatigue testing in silico can now be used to explore damage-induced changes in the material properties of other biological particles. STATEMENT OF SIGNIFICANCE: Biological particles possess mechanical characteristics necessary to perform their functions. We developed "fatigue testing in silico" approach, which employes Langevin Dynamics simulations of constant-amplitude cyclic loading of nanoscale biological particles, to explore dynamic evolution of the mechanical, energetic, and material properties of the thin and thick spherical particles of encapsulin and Cowpea Chlorotic Mottle Virus, and the microtubule filament fragment. Our study of damage growth and fatigue development challenge the existing paradigm. Damage in biological particles is partially reversible as fatigue crack might heal with each loading cycle. Particles adapt to deformation amplitude and frequency to minimize energy dissipation. The evolution of strength, deformability, and stiffness, can be accurately predicted by analyzing the damage growth in particle structure.

Therapeutic phosphorodiamidate morpholino oligonucleotides: Physical properties, solution structures, and folding thermodynamics.

Elucidating the structure-function relationships for therapeutic RNA mimicking phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to the lack of information about their structures. While PMOs have been approved by the US Food and Drug Administration for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge-neutral, and stable molecules is available. We performed circular dichroism and solution viscosity measurements combined with molecular dynamics simulations and machine learning to resolve solution structures of 22-mer, 25-mer, and 30-mer length PMOs. The PMO conformational dynamics are defined by the competition between non-polar nucleobases and uncharged phosphorodiamidate groups for shielding from solvent exposure. PMO molecules form non-canonical, partially helical, stable folded structures with a small 1.4- to 1.7-nm radius of gyration, low count of three to six base pairs and six to nine base stacks, characterized by -34 to -51 kcal/mol free energy, -57 to -103 kcal/mol enthalpy, and -23 to -53 kcal/mol entropy for folding. The 4.5- to 6.2-cm3/g intrinsic viscosity and Huggins constant of 4.5-9.9 are indicative of extended and aggregating systems. The results obtained highlight the importance of the conformational ensemble view of PMO solution structures, thermodynamic stability of their non-canonical structures, and concentration-dependent viscosity properties. These principles form a paradigm to understand the structure-properties-function relationship for therapeutic PMOs to advance the design of new RNA-mimic-based drugs.

Biomechanics, Energetics, and Structural Basis of Rupture of Fibrin Networks.

Fibrin provides the main structural integrity and mechanical strength to blood clots. Failure of fibrin clots can result in life-threating complications, such as stroke or pulmonary embolism. The dependence of rupture resistance of fibrin networks (uncracked and cracked) on fibrin(ogen) concentrations in the (patho)physiological 1-5 g L-1 range is explored by performing the ultrastructural studies and theoretical analysis of the experimental stress-strain profiles available from mechanical tensile loading assays. Fibrin fibers in the uncracked network stretched evenly, whereas, in the cracked network, fibers around the crack tip showed greater deformation. Unlike fibrin fibers in cracked networks formed at the lower 1-2.7 g L-1 fibrinogen concentrations, fibers formed at the higher 2.7-5 g L-1 concentrations align and stretch simultaneously. Cracked fibrin networks formed in higher fibrinogen solutions are tougher yet less extensible. Statistical modeling revealed that the characteristic strain for fiber alignment, crack size, and fracture toughness of fibrin networks control their rupture resistance. The results obtained provide a structural and biomechanical basis to quantitatively understand the material properties of blood plasma clots and to illuminate the mechanisms of their rupture.

Charge transport in individual short base stacked single-stranded RNA molecules.

Charge transport in biomolecules is crucial for many biological and technological applications, including biomolecular electronics devices and biosensors. RNA has become the focus of research because of its importance in biomedicine, but its charge transport properties are not well understood. Here, we use the Scanning Tunneling Microscopy-assisted molecular break junction method to measure the electrical conductance of particular 5-base and 10-base single-stranded (ss) RNA sequences capable of base stacking. These ssRNA sequences show single-molecule conductance values around [Formula: see text] ([Formula: see text]), while equivalent-length ssDNAs result in featureless conductance histograms. Circular dichroism (CD) spectra and MD simulations reveal the existence of extended ssRNA conformations versus folded ssDNA conformations, consistent with their different electrical behaviors. Computational molecular modeling and Machine Learning-assisted interpretation of CD data helped us to disentangle the structural and electronic factors underlying CT, thus explaining the observed electrical behavior differences. RNA with a measurable conductance corresponds to sequences with overall extended base-stacking stabilized conformations characterized by lower HOMO energy levels delocalized over a base-stacking mediating CT pathway. In contrast, DNA and a control RNA sequence without significant base-stacking tend to form closed structures and thus are incapable of efficient CT.

Mechanical transition from α-helical coiled coils to ß-sheets in fibrin(ogen).

We characterized the α-to-ß transition in α-helical coiled-coil connectors of the human fibrin(ogen) molecule using biomolecular simulations of their forced elongation and theoretical modeling. The force (F)-extension (X) profiles show three distinct regimes: (1) the elastic regime, in which the coiled coils act as entropic springs (F < 100-125 pN; X < 7-8 nm); (2) the constant-force plastic regime, characterized by a force-plateau (F ≈ 150 pN; X ≈ 10-35 nm); and (3) the nonlinear regime (F > 175-200 pN; X > 40-50 nm). In the plastic regime, the three-stranded α-helices undergo a noncooperative phase transition to form parallel three-stranded ß-sheets. The critical extension of the α-helices is 0.25 nm, and the energy difference between the α-helices and ß-sheets is 4.9 kcal/mol per helical pitch. The soft α-to-ß phase transition in coiled coils might be a universal mechanism underlying mechanical properties of filamentous α-helical proteins.

Single-molecule conductance of double-stranded RNA oligonucleotides.

RNA oligonucleotides are crucial for a range of biological functions and in many biotechnological applications. Herein, we measured, for the first time, the conductance of individual double-stranded (ds)RNA molecules and compared it with the conductance of single DNA : RNA hybrids. The average conductance values are similar for both biomolecules, but the distribution of conductance values shows an order of magnitude higher variability for dsRNA, indicating higher molecular flexibility of dsRNA. Microsecond Molecular Dynamics simulations explain this difference and provide structural insights into the higher stability of DNA : RNA duplex with atomic level of detail. The rotations of 2'-OH groups of the ribose rings and the bases in RNA strands destabilize the duplex structure by weakening base stacking interactions, affecting charge transport, and making single-molecule conductance of dsRNA more variable (dynamic disorder). The results demonstrate that a powerful combination of state-of-the-art biomolecular electronics techniques and computational approaches can provide valuable insights into biomolecules' biophysics with unprecedented spatial resolution.