Design principles governing the motility of myosin V

The molecular motor myosin V (MyoV) exhibits a wide repertoire of pathways during the stepping process, which is intimately connected to its biological function. The best understood of these is the hand-over-hand stepping by a swinging lever arm movement toward the plus end of actin filaments. Single-molecule experiments have also shown that the motor “foot stomps,” with one hand detaching and rebinding to the same site, and back-steps under sufficient load. The complete taxonomy of MyoV’s load-dependent stepping pathways, and the extent to which these are constrained by motor structure and mechanochemistry, are not understood. Using a polymer model, we develop an analytical theory to describe the minimal physical properties that govern motor dynamics. We solve the first-passage problem of the head reaching the target-binding site, investigating the competing effects of backward load, strain in the leading head biasing the diffusion in the direction of the target, and the possibility of preferential binding to the forward site due to the recovery stroke. The theory reproduces a variety of experimental data, including the power stroke and slow diffusive search regimes in the mean trajectory of the detached head, and the force dependence of the forward-to-backward step ratio, run length, and velocity. We derive a stall force formula, determined by lever arm compliance and chemical cycle rates. By exploring the MyoV design space, we predict that it is a robust motor whose dynamical behavior is not compromised by reasonable perturbations to the reaction cycle and changes in the architecture of the lever arm.Myosin V (MyoV), a cytoskeletal motor protein belonging to the myosin superfamily (1), converts energy from ATP hydrolysis into the transport of intracellular cargo, such as mRNA and organelles along actin filaments (2). In its dimeric form, the motor has two actin-binding, ATPase heads connected to α-helical lever arm domains stiffened by attached calmodulins or essential light chains (Fig. 1). The nucleotide-driven mechanochemical cycle of the heads produces two changes in the lever arm orientation: a power stroke, where an actin-bound head swings the lever arm forward toward the plus (barbed) end of the filament, and a recovery stroke, which returns the arm to its original configuration when the head is detached from actin (3). The motor translates these changes into processive plus end-directed movement (4–6). By alternating head detachment, MyoV walks hand-over-hand (7, 8), taking one step of for each ATP consumed (9). At small loads, the motor can complete forward steps before dissociating from actin (6, 10, 11). Such a high unidirectional processivity requires coordination in the detachment of the two heads, a “gating” mechanism, which is believed to arise from the strain within the molecule when both heads are bound to actin (12–15). Sufficiently large opposing loads can counteract the plus end-directed bias, resulting in an increase in the probability of back-stepping (16) until the motor velocity goes to zero at a stall force of pN (4, 12, 16–19). Although MyoV is among the most extensively studied motor proteins, improvements in experimental resolution continue to provide new and surprising insights into the details of its dynamics. A beautiful recent example is the high-speed atomic force microscopy (AFM) of Kodera et al. (20), which was used to visualize not only the expected hand-over-hand stepping but additional, less well-understood processes like “foot stomping” (21, 22), where one head detaches and rebinds to the same site. Thus, a comprehensive picture of MyoV motility needs to account for all the kinetic pathways, including back-stepping and foot stomping, how they vary under load, and their relationship to the structural and chemical parameters of the motor.Open in a separate windowFig. 1.(A) Orientational states of the MyoV head with respect to its lever arm. For clarity, only one leg of the two-legged motor is shown, although the states are the same for both legs. (Left) Pr. (Middle) Po. For each state, the relaxed orientation (in the absence of tension on the lever arm end) is marked by a dashed red line. (Right) Po state with backward tension on the arm end, causing the lever arm to bend backward away from its relaxed direction. (B) Coarse-grained polymer representation of MyoV. (C) Schematic view of four MyoV kinetic pathways. For simplicity, the nucleotide-free state, following ADP release from the TH and before ATP binding, is not shown. (D) Probability of each kinetic pathway as a function of backward force F (with ) calculated from the theory using the parameter set in 22–25). The large persistence length l_p of the lever arms (26–28) allows us to propose a coarse-grained polymer model for the reaction-diffusion problem, which, in turn, yields approximate analytical expressions for all the physical observables, including binding times, run length, velocity, and stall force. We have built on the insights of earlier theoretical works (28–34), which focused on modeling a reaction network of discrete states in the mechanochemical cycle of the motor heads. Our work supplements the reaction network with an explicit treatment of the diffusive search, which has been studied using insightful Brownian dynamics simulations of forward stepping in MyoV (35). An important aspect of our theory is that it allows us to tackle not just forward steps but the full complexity of foot stomping and back-stepping across the entire force spectrum up to the stall point. In our framework, the load dependence of the MyoV behavior enters naturally, because pulling on the molecule shifts the speed and likelihood of the detached head reaching the forward or backward binding site. The competition between the time scales of first passage to the sites, and how they compare with the detachment rates of the heads, determines the partitioning of the kinetic pathways. Significantly, polymer theory gives us a direct connection between the kinetics and the structural features of the motor, like the bending elasticity of the lever arms and the orientational bias due to the power stroke. The result is a theory with only three fitting parameters that have not been previously determined through experiment, all of which have simple physical interpretations. The theoretical fit quantitatively reproduces a variety of experimental data, like the time-dependent mean trajectories of the detached head (23) and the force dependence of the backward-to-forward step ratio (16) and run length/velocity (4, 16–18, 36). We also explore more broadly the design space of MyoV structural parameters, allowing us to predict the essential requirements for the observed dynamical behavior and to answer the following questions. Is the structure of the motor dictated by certain natural constraints? How robust is the motility of MyoV to perturbations in the parameters? What are the relative contributions of head chemistry (resulting from changes in the nucleotide states) and the structural features to the measured stall force? The answers to these questions, which are provided in terms of phase diagrams, lead to testable predictions.     

Larvicidal activity of green synthesized silver nanoparticles using bark aqueous extract of Ficus racemosa against Culex quinquefasciatus and Culex gelidus

ObjectiveTo investigate the larvicidal activity of synthesized silver nanoparticles (Ag NPs) utilizing aqueous bark extract of Ficus racemosa (F. racemosa) was tested against fourth instar larvae of filariasis vector, Culex quinquefasciatus (Cx. quinquefasciatus) and japanese encephalitis vectors, Culex gelidus (Cx. gelidus).MethodsThe synthesized Ag NPs was characterized by UV-vis spectrum, X-ray diffraction (XRD), Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). The larvicidal activities were assessed for 24 h against the larvae of Cx. quinquefasciatus and Cx. gelidus with varying concentrations of aqueous bark extract of F. racemosa and synthesized Ag NPs. LC₅₀ and r² values were calculated.ResultsThe maximum efficacy was observed in crude aqueous extract of F. racemosa against the larvae of Cx. quinquefasciatus and Cx. gelidus (LC₅₀=67.72 and 63.70 mg/L; r²=0.995 and 0.985) and the synthesized Ag NPs (LC₅₀=12.00 and 11.21 mg/L; r²=0.997 and 0.990), respectively. Synthesized Ag NPs showed the XRD peaks at 2 θ values of 27.61, 29.60, 35.48, 43.48 and 79.68 were identified as (210), (121), (220), (200) and (311) reflections, respectively. The FTIR spectra of Ag NPs exhibited prominent peaks at 3 425, 2 878, 1 627 and 1 382 in the region 500-3 000 cm^?1. The peaks correspond to the presence of a stretching vibration of (NH) C=O group. SEM analysis showed shape in cylindrical, uniform and rod with the average size of 250.60 nm.ConclusionsThe biosynthesis of silver nanoparticles using bark aqueous extract of F. racemosa and its larvicidal activity against the larvae of disease spreading vectors. The maximum larvicidal efficacy was observed in the synthesized Ag NPs.     

Heavier molecular weight ocular viscoelastic devices and timing of post-operative review following cataract surgery

Background  

To assess the safety of abandoning the next day post-operative review in preference for assessment only 2 hours post-surgery for both phacoemulsification and extracapsular surgery with heavier molecular weight ocular viscoelastic devices (OVD).     

Kinetics and thermodynamics of folding in model proteins.

Monte Carlo simulations on a class of lattice models are used to probe the thermodynamics and kinetics of protein folding. We find two transition temperatures: one at T theta, when chains collapse from a coil to a compact phase, and the other at Tf (< T theta), when chains adopt a conformation corresponding to their native state. The kinetics are probed by several correlation functions and are interpreted in terms of the underlying energy landscape. The transition from the coil to the native state occurs in three distinct stages. The initial stage corresponds to a random collapse of the protein chain. At intermediate times tau c, during which much of the native structure is acquired, there are multiple pathways. For longer times tau r (>> tau c) the decay is exponential, suggestive of a late transition state. The folding time scale (approximately tau r) varies greatly depending on the model. Implications of our results for in vitro folding of proteins are discussed.     

Effect of Solanum surattense seed on the oxidative potential of cauda epididymal spermatozoa

Objective

To evaluate the effect of aqueous seed extract of Solanum surattense (S. surattense) on the oxidative potential of cauda epididymal spermatozoa.

Methods

S. surattense seed extract was orally administered at the dosage of 10 mg/kg b.w. for 15 days, after which aspartate transferase (AST), alanine transferase (ALT), glutamate dehydrogenase (GDH), citric acid and iso-citrate dehydrogenase (ICDH) were assayed.

Results

The activity levels of the enzymes AST and ALT, which are considered to be the androgenicity in the sperm suspension, were depleted in the extract fed rats. The activity level of the enzyme ICDH, was reduced significantly in the treated group (P<0.001).

Conclusions

It can be concluded that the oral administration of the aqueous seed extract of S. surattense can deplete the oxidative stress of cauda epididymal spermatozoa in albino rats.     

The IL-21 receptor augments Th2 effector function and alternative macrophage activation

The IL-21 receptor (IL-21R) shows significant homology with the IL-4R, and CD4+ Th2 cells are an important source of IL-21. Here we examined whether the IL-21R regulates the development of Th2 responses in vivo. To do this, we infected IL-21R-/- mice with the Th2-inducing pathogens Schistosoma mansoni and Nippostrongylus brasiliensis and examined the influence of IL-21R deficiency on the development of Th2-dependent pathology. We showed that granulomatous inflammation and liver fibrosis were significantly reduced in S. mansoni-infected IL-21R-/- mice and in IL-21R+/+ mice treated with soluble IL-21R-Fc (sIL-21R-Fc). The impaired granulomatous response was also associated with a marked reduction in Th2 cytokine expression and function, as evidenced by the attenuated IL-4, IL-13, AMCase, Ym1, and FIZZ1 (also referred to as RELMalpha) responses in the tissues. A similarly impaired Th2 response was observed following N. brasiliensis infection. In vitro, IL-21 significantly augmented IL-4Ralpha and IL-13Ralpha1 expression in macrophages, resulting in increased FIZZ1 mRNA and arginase-1 activity following stimulation with IL-4 and IL-13. As such, these data identify the IL-21R as an important amplifier of alternative macrophage activation. Collectively, these results illustrate an essential function for the IL-21R in the development of pathogen-induced Th2 responses, which may have relevance in therapies for both inflammatory and chronic fibrotic diseases.     

Low-frequency normal modes that describe allosteric transitions in biological nanomachines are robust to sequence variations

By representing the high-resolution crystal structures of a number of enzymes using the elastic network model, it has been shown that only a few low-frequency normal modes are needed to describe the large-scale domain movements that are triggered by ligand binding. Here we explore a link between the nearly invariant nature of the modes that describe functional dynamics at the mesoscopic level and the large evolutionary sequence variations at the residue level. By using a structural perturbation method (SPM), which probes the residue-specific response to perturbations (or mutations), we identify a sparse network of strongly conserved residues that transmit allosteric signals in three structurally unrelated biological nanomachines, namely, DNA polymerase, myosin motor, and the Escherichia coli chaperonin. Based on the response of every mode to perturbations, which are generated by interchanging specific sequence pairs in a multiple sequence alignment, we show that the functionally relevant low-frequency modes are most robust to sequence variations. Our work shows that robustness of dynamical modes at the mesoscopic level is encoded in the structure through a sparse network of residues that transmit allosteric signals.