Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release.

Inherited mutations in human beta-cardiac myosin (M2ß) can lead to severe forms of heart failure. The E525K mutation in M2ß is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2ß subfragment 1 (S1) we found that E525K enhances (3-fold) the maximum steady-state actin-activated ATPase activity (kcat) and decreases (6-fold) the actin concentration at which ATPase is one-half maximal (KATPase). We also found a 3 to 4-fold increase in the actin-activated power stroke and phosphate release rate constants at 30 µM actin, which overall enhanced the duty ratio 3-fold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2ß S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt-bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt-bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Membrane remodeling properties of the Parkinson's disease protein LRRK2.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.

Single-Molecule Manipulation Study of Chaperoned SNARE Folding and Assembly with Optical Tweezers.

Intracellular membrane fusion is primarily driven by coupled folding and assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE assembly is intrinsically inefficient and must be chaperoned by a family of evolutionarily and structurally conserved Sec1/Munc-18 (SM) proteins. The physiological pathway of the chaperoned SNARE assembly has not been well understood, partly due to the difficulty in dissecting the many intermediates and pathways of SNARE assembly and measure their associated energetics and kinetics. Optical tweezers have proven to be a powerful tool to characterize the intermediates involved in the chaperoned SNARE assembly. Here, we demonstrate the application of optical tweezers combined with a homemade microfluidic system into studies of synaptic SNARE assembly chaperoned by their cognate SM protein Munc18-1. Three synaptic SNAREs and Munc18-1 constitute the core machinery for synaptic vesicle fusion involved in neurotransmitter release. Many other proteins further regulate the core machinery to enable fusion at the right time and location. The methods described here can be applied to other proteins that regulate SNARE assembly to control membrane fusion involved in numerous biological and physiological processes.

Quantitative Models of Lipid Transfer and Membrane Contact Formation.

Lipid transfer proteins (LTPs) transfer lipids between different organelles, and thus play key roles in lipid homeostasis and organelle dynamics. The lipid transfer often occurs at the membrane contact sites (MCS) where two membranes are held within 10-30 nm. While most LTPs act as a shuttle to transfer lipids, recent experiments reveal a new category of eukaryotic LTPs that may serve as a bridge to transport lipids in bulk at MCSs. However, the molecular mechanisms underlying lipid transfer and MCS formation are not well understood. Here, we first review two recent studies of extended synaptotagmin (E-Syt)-mediated membrane binding and lipid transfer using novel approaches. Then we describe mathematical models to quantify the kinetics of lipid transfer by shuttle LTPs based on a lipid exchange mechanism. We find that simple lipid mixing among membranes of similar composition and/or lipid partitioning among membranes of distinct composition can explain lipid transfer against a concentration gradient widely observed for LTPs. We predict that selective transport of lipids, but not membrane proteins, by bridge LTPs leads to osmotic membrane tension by analogy to the osmotic pressure across a semipermeable membrane. A gradient of such tension and the conventional membrane tension may drive bulk lipid flow through bridge LTPs at a speed consistent with the fast membrane expansion observed in vivo. Finally, we discuss the implications of membrane tension and lipid transfer in organelle biogenesis. Overall, the quantitative models may help clarify the mechanisms of LTP-mediated MCS formation and lipid transfer.

Single-Molecule Optical Tweezers Study of Protein-Membrane Interactions.

Numerous proteins directly or indirectly bind membranes to exert their roles in a wide variety of biological processes. Such membrane binding often occurs in the presence of an external mechanical force. It remains challenging to quantify these interactions using traditional experimental approaches based on a large number of molecules, due to ensemble averaging or the lack of mechanical force. Here we described a new single-molecule approach based on high-resolution optical tweezers to characterize protein-membrane interactions. A single membrane binding protein is attached to the lipid bilayer coated on a silica bead via a flexible polypeptide linker, tethered to another bead via a long DNA handle, and pulled away from the bilayer using optical tweezers. Dynamic protein binding and unbinding is detected by the corresponding changes in the extension of the protein-DNA tether with high spatiotemporal resolution, which reveals the membrane binding affinity, kinetics, and intermediates. We demonstrated the method using C2 domains of extended synaptotagmin 2 (E-Syt2) with a detailed protocol. The method can be widely applied to investigate complex protein-membrane interactions under well-controlled experimental conditions.

Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers.

Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin.

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human ß-cardiac myosin subfragment 1 (M2ß-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2ß-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2ß-S1. Our results demonstrated M2ß-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2ß-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2ß-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.

CO2 Laser Moxibustion for Knee Osteoarthritis: Study Protocol for A Multicenter, Double-blind, Randomized Controlled Trial.

BACKGROUND: Knee osteoarthritis (OA) is a major cause of disability among the older adults. Few treatments are safe and effective. Moxibustion is commonly used in treating knee OA in Chinese medicine (CM). CO2 Laser moxibustion device is a substitute for traditional moxibustion, which mimics the effects of traditional moxibustion. More data are needed to support its application in knee OA. OBJECTIVE: ObjectiveThe trial aims to assess the effect and safety of CO2 laser moxibustion in patients with knee osteoarthritis compared with a sham control. METHODS: This is a protocol for a multicenter, randomized, double-blind, placebo-controlled trial. A total of 392 participants were recruited and assigned to the CO2 laser moxibustion group and sham laser moxibustion group with a 1:1 ratio at 6 outpatient clinics in Shanghai, China. Participants in both groups received treatment at the affected knee(s) at the acupuncture point Dubi (ST 35) and an Ashi point. There were 3 sessions per week for 4 weeks, and an additional 20-week follow-up. Primary outcomes were changes in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain scores at week 4. Secondary outcomes were WOMAC function score, stiffness score and overall score, VAS pain, Short-Form heath survey (SF-36), and patients' global assessment. The serum levels of cytokines involved in progress of knee OA were explored. Safety was assessed during the whole trial. Masking effectiveness was assessed by both participants and treatment providers.This is a protocol for a multicenter, randomized, double-blind, placebo-controlled trial. A total of 392 participants were recruited and assigned to the CO2 laser moxibustion group and sham laser moxibustion group with a 1:1 ratio at 6 outpatient clinics in Shanghai, China. Participants in both groups received treatment at the affected knee(s) at the acupuncture point Dubi (ST 35) and an Ashi point. There were 3 sessions per week for 4 weeks, and an additional 20-week follow-up. Primary outcomes were changes in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain scores at week 4. Secondary outcomes were WOMAC function score, stiffness score and overall score, VAS pain, Short-Form heath survey (SF-36), and patients' global assessment. The serum levels of cytokines involved in progress of knee OA were explored. Safety was assessed during the whole trial. Masking effectiveness was assessed by both participants and treatment providers. DISCUSSION: CO2 laser moxibustion device, designed as a substitute for CM moxibustion, is easy to use and control with no choking smoke and smell, and is a plausible method for double-blind research. This study would provide rigorous evidence for the effect and safety of CO2 laser moxibustion in treating knee OA (Trial registration No.: ISRCTN15030019).

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke.

Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

Electrostatic interactions in the force-generating region of the human cardiac myosin modulate ADP dissociation from actomyosin.

Human cardiac myosin has two isoforms, alpha and beta, sharing significant sequence similarity, but different in kinetics: ADP release from actomyosin is an order of magnitude faster in the alpha myosin isoform. Apparently, small differences in the sequence are responsible for distinct local inter-residue interactions within alpha and beta isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of alpha and beta isoforms using molecular dynamics simulations revealed distinct dynamics of SH1:SH2 helix within the force-generation region of myosin head. The simulations showed that the residue R694 of the helix forms two permanent salt bridges in the beta isoform, which are not present in the alpha isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N mutant in the beta isoform background to destabilize electrostatic interactions in the force-generating region of the myosin head. Our experimental data confirm faster ADP release from R694N actomyosin mutant, but is not as dramatic as the difference of kinetics of ADP release in the alpha and beta isoforms.

Metal cation controls phosphate release in the myosin ATPase.

Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium "holds" phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin-nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate.

Macromolecular Crowding Modulates Actomyosin Kinetics.

Actomyosin kinetics is usually studied in dilute solutions, which do not reflect conditions in the cytoplasm. In cells, myosin and actin work in a dense macromolecular environment. High concentrations of macromolecules dramatically reduce the amount of free space available for all solutes, which results in an effective increase of the solutes' chemical potential and protein stabilization. Moreover, in a crowded solution, the chemical potential depends on the size of the solute, with larger molecules experiencing a larger excluded volume than smaller ones. Therefore, since myosin interacts with two ligands of different sizes (actin and ATP), macromolecular crowding can modulate the kinetics of individual steps of the actomyosin ATPase cycle. To emulate the effect of crowding in cells, we studied actomyosin cycle reactions in the presence of a high-molecular-weight polymer, Ficoll70. We observed an increase in the maximum velocity of the actomyosin ATPase cycle, and our transient-kinetics experiments showed that virtually all individual steps of the actomyosin cycle were affected by the addition of Ficoll70. The observed effects of macromolecular crowding on the myosin-ligand interaction cannot be explained by the increase of a solute's chemical potential. A time-resolved Förster resonance energy transfer experiment confirmed that the myosin head assumes a more compact conformation in the presence of Ficoll70 than in a dilute solution. We conclude that the crowding-induced myosin conformational change plays a major role in the changed kinetics of actomyosin ATPase.

Analysis of Flame Retardancy in Polymer Blends by Synchrotron X-ray K-edge Tomography and Interferometric Phase Contrast Movies.

Underwriters Laboratories 94 test bars have been imaged with X-ray K-edge tomography between 12 and 32 keV to assess the bromine and antimony concentration gradient across char layers of partially burnt samples. Phase contrast tomography on partially burnt samples showed gas bubbles and dark-field scattering ascribed to residual blend inhomogeneity. In addition, single-shot grating interferometry was used to record X-ray movies of test samples during heating (IR and flame) intended to mimic the UL 94 plastics flammability test. The UL 94 test bars were formulated with varying concentrations of a brominated flame retardant, Saytex 8010, and a synergist, Sb2O3, blended into high-impact polystyrene (HIPS). Depending on the sample composition, samples will pass or fail the UL 94 plastics flammability test. Tomography and interferometry imaging show differences that correlate with UL 94 performance. Key features such as char layer, gas bubble formation, microcracks, and dissolution of the flame retardant in the char layer regions are used in understanding the efficiency of the flame retardant and synergist. The samples that pass the UL 94 test have a thick, highly visible char layer as well as an interior rich in gas bubbles. Growth of gas bubbles from flame-retardant thermal decomposition is noted in the X-ray phase contrast movies. Also noteworthy is an absence of bubbles near the burning surface of the polymer; dark-field images after burning suggest a microcrack structure between interior bubbles and the surface. The accepted mechanism for flame retardant activity includes free radical quenching in the flame by bromine and antimony species. The imaging supports this as well as provides a fast inspection of other parameters, such as viscosity and surface tension.

Metal cation controls myosin and actomyosin kinetics.

We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time-resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.