Search

The identical structural interaction between phospholamban and Ca-ATPase and the functional inhibition indicate that the inhibitory regulation of the enzyme is mediated by alpha-helices stabilization. Spectral shifts in both amide I and II bands and circular dichroism, upon co-reconstitution of the enzyme with phospholamban, is indicative of alpha-helical stability.

Research demonstrates that phospholamban exists as a homopentamer and self-association forms oligomers. The protein kinase activation of phospholambans is a required step for the beta-adrenergic stimulation of the calcium-ATPase upon reconstitution into proteoliposomes.

Engineered phospholamban (PLB) mutant is used for identifying the conformational changes in the hinge structure of the cystosolic portion of PLB, which may be a result of the phosphorylation of cAMP-dependent protein kinase (PKA) or calcium activation of the Ca-ATPase. The conformational constraints imposed by PLB association with the nucleotide-binding domain of the Ca-ATPase can overcome due to the calcium-binding to the Ca-ATPase.

The phospholipase A 2 (PLA 2 ) superfamily consists of lipolytic enzymes that hydrolyze specific cell membrane phospholipids and have long been considered a central hub of biosynthetic pathways, where their lipid metabolites exert a variety of physiological roles. A misregulated PLA 2 activity is associated with mainly inflammatory-derived pathologies and thus has shown relevant therapeutic potential. Many natural and synthetic anti-inflammatory drugs (AIDs) have been proposed as direct modulators of PLA 2 activity. However, despite the specific chemical properties that these drugs share in common, little is known about the indirect modulation able to finely tune membrane structural changes at the precise lipid-binding site. Here, we use a novel experimental strategy based on differential scanning calorimetry to systematically study the structural properties of lipid membrane systems during PLA 2 cleavage and under the influence of several AIDs. For a better understanding of the AIDs-membrane interaction, we present a comprehensive and comparative set of molecular dynamics (MD) simulations. Our thermodynamic results clearly demonstrate that PLA 2 cleavage is hindered by those AIDs that significantly reduce the lipid membrane cooperativity, while the rest of the AIDs oppositely tend to catalyze PLA 2 activity to different extents. On the other hand, our MD simulations support experimental results by providing atomistic details on the binding, insertion, and dynamics of each AID on a pure lipid system; the drug efficacy to impact membrane cooperativity is related to the lipid order perturbation. This work suggests a membrane-based mechanism of action for diverse AIDs against PLA 2 activity and provides relevant clues that must be considered in its modulation.

Cobalt-free (Co-free) and nickel-rich (Ni-rich) cathode materials have attracted significant attention and undergone extensive studies due to their affordability and superior energy density. However, the commercialization of these Co-free materials is hindered by challenges such as cation disorder, irreversible phase changes, and inadequate high-voltage performance. To overcome these challenges, a Co-free ternary cathode material of Mg/Al double-pillared LiNiO 2 (NMA) synthesized via a wet-coating and lithiation-sintering technique is proposed. Fundamental studies reveal that Mg and Al have the potential to form a distinctive double-pillar structure within the layered cathode, enhancing its structural stability. To be specific, the strategic placement of Mg and Al in Li and Ni layers, respectively, effectively reduces Li + /Ni 2+ disorder and prevents irreversible phase transitions. Additionally, the inclusion of Mg and Al refines the primary grains and compacts the secondary grains in the cathode material, reducing stress from cyclic usage and preventing material cracking, thereby mitigating electrolyte erosion. As a result, NMA demonstrates exceptional electrochemical performance under a high charge cutoff voltage of 4.6 V. It maintains 70% of initial specific capacity after 500 cycles at 1 C and exhibits excellent rate performance, with a capacity of 162 mAh g -1 at 5 C and 149 mAh g -1 at 10 C. As a whole, the produced NMA achieves a high structural stability in cases of excessive delithiation, providing a groundbreaking solution for the development of cost-effective and high-energy-density cathode materials for lithium-ion batteries.

This comprehensive molecular dynamics (MD) simulation and experimental study investigates the lipid bilayer interactions of dye D112 for potential photodynamic therapy (PDT) applications. PDT involves formation of a reactive oxidant species in the presence of a light sensitive molecule and light, interrupting cellular functions. D112 was developed as a photographic emulsifier, and we hypothesized that its combined cationic and lipophilic nature can render a superior photosensitizing property-crucial in various light therapies. The focus of this study is to elucidate the binding and insertion mechanisms of D112 with mixed lipid bilayers of anionic dipalmitoyl-phosphatidylserine (DPPS) and zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) lipids to resemble cancer cell membranes. Our studies confirm initial electrostatic binding between the positively charged moieties of D112 and negatively charged lipid headgroups. Additionally, MD simulations combined with differential scanning calorimetry (DSC) studies confirm that D112-lipid interactions are governed by enthalpy-driven nonclassical hydrophobic effects in the membrane interior. It was further noted that despite the electrostatic preference of D112 toward the anionic lipids, D112 molecules colocalized on DPPC-rich domains after insertion. Atomistic level MD studies point toward two possible insertion mechanisms for D112: harpoon and flip. Further insights from the simulation showcase the interactions of low and high aggregates of D112 with the bilayer as the concentration of D112 increases in solution. The size of aggregates modulates the orientation and degree of insertion, providing important information for future studies on membrane permeation mechanisms., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Unclear impurity pollution is one of the key scientific problems that limit the large-scale production of new lithium-ion batteries (LIBs) from spent LIBs. This work is the first to report the pollution path, pollution degree, and solution method of sodium ions in the recycling process of spent LIBs in the real world. The results show that sodium ions can intrude into the precursor particles to form crystalline salts with the anion of the leaching acid that cover the transition metal elements, thereby resulting in a failed precursor. Specifically, the intrusion of sodium ions will produce a variety of pollutants containing metal oxide bonds, such as Na-O, NaO 2 , and Na + -O 2 , on the precursor surface. These active lattice oxygen will further adsorb or react to form organic oxygen, chemical oxygen, and free oxygen, which will highly deteriorate the surface cleanliness. Strictly controlling the consumption of sodium salt in each step and using ammonia instead of NaOH for pH regulation can effectively solve sodium ion pollution to prepare high-quality battery precursors. It reveals that for the green upcycling of spent LIBs, we should strengthen the design of the recycling process to reduce the consumption of chemical reagents, which will produce unexpected secondary pollution.

Lithium (Li) is the most valuable metal in spent lithium iron phosphate (LiFePO 4 ) batteries, but its recovery has become a challenge in electronic waste recovery because of its relatively low content and inconsistent quality. This study proposes an acid-free and selective Li extraction process to successfully achieve the isomorphic substitution of Li in LiFePO 4 crystals with sodium (Na). The method uses low-cost and nontoxic sodium chloride (NaCl) as a cogrinding reagent via a mechanical force-induced solid-phase reaction. X-ray diffraction (XRD), energy dispersive X-ray analysis (EDAX), and X-ray photoelectron spectroscopy (XPS) characterizations demonstrated the evidence of Li/Na isomorphic substitution, while XPS spectra of Fe 2p, P 2p, and O 1s revealed that the coordination environment of these elements was not significantly altered. Density functional theory calculations further provided evidence that Na and Li share similar outer electron arrangements and coordination environments, favoring Na over Fe as a replacement for Li in LiFePO 4 . Additionally, the regeneration of NaCl and the recovery of precipitated Li 2 CO 3 were simultaneously achieved with Na 2 CO 3 as the sole reagent. This concise and efficient acid-free mechanochemical process for Li extraction is a promising candidate for feasible recycling technology of Li from spent LiFePO 4 batteries. The proposed process is particularly appealing because of its high selectivity, considerable economic advantages, and environmental benefits.

Wearable electronics with excellent stretchability and sensitivity have emerged as a very promising field with wide applications such as e-skin and human motion detection. Although three-dimensional (3D) graphene structures (GS) have been reported for high-performance strain sensors, challenges still remain such as the high cost of GS preparation, low stretchability, and the lack of ability to heal itself. In this paper, we reported a novel self-healing flexible electronics with 3D GS based on Diels-Alder (DA) chemistry. Furfurylamine (FA) was employed as a reducing as well as a modifying agent, forming GS by FA (FAGS)/DA bonds contained polyurethane with the "infiltrate-gel-dry" process. The as-prepared composite exhibited excellent stretchability (200%) and intrinsic conductivity with low incorporation of graphene (about 2 wt %), which could be directly employed for flexible electronics to detect human motions. Besides, the FAGS/DAPU composite exhibited lower temperature retro-DA response for the continuous graphene networks. Highly effective healing of the composites by heat and microwave has been demonstrated successfully.

Stretchable and flexible sensors attached onto the surface of the human body can perceive external stimuli, thus attracting extensive attention due to their lightweight, low modulus, low cost, high flexibility, and stretchability. Recently, a myriad of efforts have been devoted to improving the performance and functionality of wearable sensors. Herein, this review focuses on recent remarkable advancements in the development of flexible and stretchable sensors. Multifunction of these wearable sensors is realized by incorporating some desired features (e.g., self-healing, self-powering, linearity, and printing). Next, focusing on the characteristics of carbon nanomaterials, nanostructured metal, conductive polymer, or their hybrid composites, two major strategies (e.g., materials that stretch and structures that stretch) and diverse design approaches have been developed to achieve highly flexible and stretchable electrodes. Strain sensing performances of recently reported sensors indicate that the appropriate choice of geometric engineering as well as intrinsically stretchable materials is essential for high-performance strain sensing. Finally, some important directions and challenges of a fully sensor-integrated wearable platform are proposed to realize their potential applications for human motion monitoring and human-machine interfaces.

The continuous evolution toward flexible electronics with mechanical robust property and restoring structure simultaneously places high demand on a set of polymeric material substrate. Herein, we describe a composite material composed of a polyurethane based on Diels-Alder chemistry (PU-DA) covalently linked with functionalized graphene nanosheets (FGNS), which shows mechanical robust and infrared (IR) laser self-healing properties at ambient conditions and is therefore suitable for flexible substrate applications. The mechanical strength can be tuned by varying the amount of FGNS and breaking strength can reach as high as 36 MPa with only 0.5 wt % FGNS loading. On rupture, the initial mechanical properties are restored with more than 96% healing efficiency after 1 min irradiation time by 980 nm IR laser. Especially, this is the highest value of healing efficiency reported in the self-healable materials based on DA chemistry systems until now, and the composite exhibits a high volume resistivity up to 5.6 × 10 11 Ω·cm even the loading of FGNS increased to 1.0 wt %. Moreover, the conductivity of the broken electric circuit which was fabricated by silver paste drop-cast on the healable composite substrate was completely recovered via IR laser irradiating bottom substrate mimicking human skin. These results demonstrate that the FGNS-PU-DA nanocomposite can be used as self-healing flexible substrate for the next generation of intelligent flexible electronics.

Sorption-enhanced steam reforming (SESR) is an energy and cost efficient approach to produce hydrogen with high purity. SESR makes it economically feasible to use a wide range of feedstocks for hydrogen production such as methane, ethanol, and biomass. Selection of catalysts and sorbents plays a vital role in SESR. This article reviews the recent research aimed at process intensification by the integration of catalysis and chemisorption functions into a single material. Alkali metal ceramic powders, including Li 2 ZrO 3 , Li 4 SiO 4 and Na 2 ZrO 3 display characteristics suitable for capturing CO 2 at low concentrations (<15% CO 2 ) and high temperatures (>500 °C), and thus are applicable to precombustion technologies such as SESR, as well as postcombustion capture of CO 2 from flue gases. This paper reviews the progress made in improving the operational performance of alkali metal ceramics under conditions that simulate power plant and SESR operation, by adopting new methods of sorbent synthesis and doping with additional elements. The paper also discusses the role of carbonates formed after in situ CO 2 chemisorption during a steam reforming process in respect of catalysts for tar cracking.

Hydrogels usually suffer from low mechanical strength, which largely limit their application in many fields. In this Research Article, we prepared a dual physically cross-linked hydrogel composed of poly(acrylamide-co-acrylic acid) (PAM-co-PAA) and poly(vinyl alcohol) (PVA) by simple two-steps methods of copolymerization and freezing/thawing. The hydrogen bond-associated entanglement of copolymer chains formed as cross-linking points to construct the first network. After being subjected to the freezing/thawing treatment, PVA crystalline domains were formed to serve as knots of the second network. The hydrogels were demonstrated to integrate strength and toughness (1230 ± 90 kPa and 1250 ± 50 kJ/m(3)) by the introduction of second physically cross-linked network. What̀s more, the hydrogels exhibited rapid recovery, excellent fatigue resistance, and self-healing property. The dynamic property of the dual physically cross-linked network contributes to the excellent energy dissipation and self-healing property. Therefore, this work provides a new route to understand the toughness mechanism of dual physically cross-linked hydrogels, hopefully promoting current hydrogel research and expanding their applications.

Wearable strain sensors with excellent stretchability and sensitivity have emerged as a very promising field which could be used for human motion detection and biomechanical systems, etc. Three-dimensional (3D) graphene foam (GF) has been reported before for high-performance strain sensors, however, some problems such as high cost preparation, low sensitivity, and stretchability still remain. In this paper, we report a highly stretchable and sensitive strain sensor based on 3D GF and polydimethylsiloxane (PDMS) composite. The GF is prepared by assembly process from graphene oxide via a facile and scalable method and possesses excellent mechanical property which facilitates the infiltration of PDMS prepolymer into the graphene framework. The as-prepared strain sensor can be stretched as high as 30% of its original length and the gauge factor of this sensor is as high as 98.66 under 5% of applied strain. Moreover, the strain sensor shows long-term stability in 200 cycles of stretching-relaxing. Implementation of the device for monitoring the bending of elbow and finger results in reproducibility and various responses in the form of resistance change. Thus, the developed strain sensors exhibit great application potential in fields of biomechanical systems and human-interactive applications.

Newly defined categories of WEEE have increased the types of China's regulated WEEE from 5 to 14. Identification of the amounts and valuable-resource components of the "new" WEEE generated is critical to solving the e-waste problem, for both governmental policy decisions and recycling enterprise expansions. This study first estimates and predicts China's new WEEE generation for the period of 2010-2030 using material flow analysis and the lifespan model of the Weibull distribution, then determines the amounts of valuable resources (e.g., base materials, precious metals, and rare-earth minerals) encased annually in WEEE, and their dynamic transfer from in-use stock to waste. Main findings include the following: (i) China will generate 15.5 and 28.4 million tons WEEE in 2020 and 2030, respectively, and has already overtaken the U.S. to become the world's leading producer of e-waste; (ii) among all the types of WEEE, air conditioners, desktop personal computers, refrigerators, and washing machines contribute over 70% of total WEEE by weight. The two categories of EEE-electronic devices and electrical appliances-each contribute about half of total WEEE by weight; (iii) more and more valuable resources have been transferred from in-use products to WEEE, significantly enhancing the recycling potential of WEEE from an economic perspective; and (iv) WEEE recycling potential has been evolving from ∼16 (10-22) billion US$ in 2010, to an anticipated ∼42 (26-58) billion US$ in 2020 and ∼73.4 (44.5-103.4) billion US$ by 2030. All the obtained results can improve the knowledge base for closing the loop of WEEE recycling, and contribute to governmental policy making and the recycling industry's business development.

A number of efforts have been launched to solve the global electronic waste (e-waste) problem. The efficiency of e-waste recycling is subject to variable national legislation, technical capacity, consumer participation, and even detoxification. E-waste management activities result in procedural irregularities and risk disparities across national boundaries. We review these variables to reveal opportunities for research and policy to reduce the risks from accumulating e-waste and ineffective recycling. Full regulation and consumer participation should be controlled and reinforced to improve local e-waste system. Aiming at standardizing best practice, we alter and identify modular recycling process and infrastructure in eco-industrial parks that will be expectantly effective in countries and regions to handle the similar e-waste stream. Toxicity can be deleted through material substitution and detoxification during the life cycle of electronics. Based on the idea of "Control-Alt-Delete", four patterns of the way forward for global e-waste recycling are proposed to meet a variety of local situations.

Recycling metals from wastes is essential to a resource-efficient economy, and increasing attention from researchers has been devoted to this process in recent years, with emphasis on mechanochemistry technology. The mechanochemical method can make technically feasible the recycling of metals from some specific wastes, such as cathode ray tube (CRT) funnel glass and tungsten carbide waste, while significantly improving recycling efficiency. Particle size reduction, specific surface area increase, crystalline structure decomposition and bond breakage have been identified as the main processes occurring during the mechanochemical operations in the studies. The activation energy required decreases and reaction activity increases, after these changes with activation progress. This study presents an overall review of the applications of mechanochemistry to metal recycling from wastes. The reaction mechanisms, equipment used, method procedures, and optimized operating parameters of each case, as well as methods enhancing the activation process are discussed in detail. The issues to be addressed and perspectives on the future development of mechanochemistry applied for metal recycling are also presented.

Coassemble diverse functional nanomaterials with carbon nanotubes (CNTs) to form three-dimensional (3D) porous CNTs hybrid architectures (CHAs) are potentially desirable for applications in energy storage, flexible conductors, and catalysis, because of diverse functionalities and synergistic effects in the CHAs. Herein, we report a scalable strategy to incorporate various functional nanomaterials with N-doped CNTs (N-CNTs) into such 3D porous CHAs on the polyurethane (PU) sponge skeletons via layer-by-layer (LbL) assembly. To investigate their properties and applications, the specific CHAs based on N-CNTs and Ag nanoparticles (NPs), denoted as PU-(N-CNTs/Ag NPs)n, are developed. The unique binary structure enables these specific CHAs conductors to possess reliable mechanical and electrical performance under various elastic deformations as well as excellent hydrophilicity. Moreover, they are employed as strain-gauge sensor and heterogeneous catalyst, respectively. The sensor could detect continuous signal, static signal, and pulse signal with superior sustainability and reversibility, indicating an important branch of electromechanical devices. Furthermore, the synergistic effects among N-CNTs, Ag NPs, and porous structure endow the CHAs with excellent performance in catalysis. We have a great expectation that LbL assembly can afford a universal route for incorporating diverse functional materials into one structure.

Flexible strain-driven sensor is an essential component in the flexible electronics. Especially, high durability and sensitivity to strain are required. Here, we present an efficient and low-cost fabrication strategy to construct a highly sensitive and flexible pressure sensor based on a conductive, elastic aerogel with pyramid design. When pressure is loaded, the contact area between the interfaces of the conductive aerogel and the copper electrode as well as among the building blocks of the nitrogen-doped carbon-nanotube-supported Ag (N-CNTs/Ag) aerogel monoliths, changes in reversible and directional manners. This contact resistance mechanism enables the hybrid aerogels to act as strain-driven sensors with high sensitivity and excellent on/off swithching behavior, and the gauge factor (GF) is ∼15 under strain of 3%, which is superior to those reported for other aerogels. In addition, robust, elastomeric and conductive nanocomposites can be fabricated by injecting polydimethylsiloxane (PDMS) into alginate/N-CNTs/Ag aerogels. Importantly, the building blocks forming the aerogels retain their initial contact and percolation after undergoing large-strain deformation, PDMS infiltration, and cross-linking of PDMS, suggesting their potential applications as strain sensors.

The disposal of scrap cathode ray tube (CRT) funnel glass has become a global environmental problem due to the rapid shrinkage of new CRT monitor demand, which greatly reduces the reuse for remanufacturing. To detoxificate CRT funnel glass by lead recovery with traditional metallurgical methods, mechanical activation by ball milling was introduced to pretreat the funnel glass. As a result, substantial physicochemical changes have been observed after mechanical activation including chemical breakage and defects formation in glass inner structure. These changes contribute to the easy dissolution of the activated sample in solution. High yield of 92.5% of lead from activated CRT funnel glass by diluted nitric acid leaching and successful formation of lead sulfide by sulfur sulfidization in water have also been achieved. All the results indicate that the application of mechanical activation on recovering lead from CRT funnel glass is efficient and promising, which is also probably appropriate to detoxificate any other kind of leaded glass.

Many developing countries have not significantly changed their course with regard to electronic waste contamination, and they are still facing the specter of mountains of hazardous electronic waste, with serious consequences for both the environment and public health. An efficient and stable analytical method was developed to determine the inventory and emission factors of polybrominated dibenzo-p-dioxin and dibenzofurans (PBDD/Fs) and polychlorinated dibenzo-p-dioxin and dibenzofurans (PCDD/Fs) formed from the incineration of scrap printed circuit boards (PCBs). Both PBDD/Fs and PCDD/Fs have been found in all experimental sections with a maximum formation rate at temperatures between 250 and 400 °C. The amounts tended first to increase and then began to decrease as the temperature rose. When subjected to a heating temperature of 325 °C, the total content of twelve 2,3,7,8-substituted PBDD/Fs congeners (tetra- through octabromo-) gathered from three outputs was the largest, at 19 000, 160 000, and 57 ng TEQ/kg in solid, liquid, and gaseous fractions, respectively; the total content of seventeen 2,3,7,8-substituted PCDD/Fs congeners (tetra- through octachloro-) was 820, 550, and 1.4 ng TEQ/kg. The formation of PCDD/Fs was remarkably less than that of PBDD/Fs because bromine concentrations considerably exceeded chlorine concentrations. The ingredients and conditions necessary to form PCDD/Fs or PBDD/Fs were definitely present, such as products of incomplete combustion, halogenides, an oxidizing atmosphere, and a catalyst-Cu salts being the most effective, significantly increasing the yields of PCDD/Fs and PBDD/Fs and decreasing the optimum temperature range.

Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence, an important research objective is to detect in situ the lever arm orientation. Single-molecule measurements are preferred because they clarify ambiguities that are unavoidable with ensemble measurements; however, detecting single molecules in the condensed tissue medium where the myosin concentration exceeds 100 muM is challenging. A myosin light chain (MLC) tagged with photoactivatable green fluorescent protein (PAGFP) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy, and PAGFP was sparsely photoactivated such that polarized fluorescence identified a single probe orientation. Several physiological states of the muscle fiber were characterized, revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight and bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where the myosin concentration is low suggests molecular crowding in the A-band promotes occupancy of the straight myosin conformation [Burghardt, T. P., et al. (2007) Biophys. J. 93, 2226]. The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke.

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.

Mutagenesis and cross-linking measurements have identified specific contact interactions between the cytosolic and the transmembrane sequences of phospholamban (PLB) and the Ca-ATPase, and in conjunction with the high-resolution structures of PLB and the Ca-ATPase, have been used to construct models of the PLB-ATPase complex, which suggest that PLB adopts a more extended structure within this complex. To directly test these predictions, we have used fluorescence resonance energy transfer to measure the average conformation and heterogeneity between chromophores covalently bound to the transmembrane and cytosolic domains of PLB reconstituted in proteoliposomes. In the absence of the Ca-ATPase, the cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, which can be described using a model involving a Gaussian distribution of distances with an average distance (Rav) of less than 21 A and a half-width (HW) of 36 A. This conformational heterogeneity of PLB is consistent with the 10 structures resolved by NMR for the C41F mutant of PLB in organic cosolvents. In contrast, PLB bound to the Ca-ATPase assumes a unique and highly ordered conformation, where Rav = 14.0 +/- 0.3 A and HW = 3.7 +/- 0.6 A. The small spatial separation between the bound chromophores on PLB is inconsistent with an extended conformation of bound PLB in current models. Thus, to satisfy known interaction sites of PLB and the Ca-ATPase, these findings suggest a reorientation of the nucleotide binding domain of the Ca-ATPase toward the bilayer surface to bring known PLB binding sites into close juxtaposition with residues near the amino-terminus of PLB. Induction of an altered conformation of the nucleotide binding domain of the Ca-ATPase by PLB binding is suggested to underlie the reduced calcium sensitivity associated with PLB inhibition of the pump.

We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys(24)) derivatized with the fluorophore N-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys(24) in the transmembrane domain and Tyr(6) in the cytosolic domain near the amino terminus of PLB. In these measurements, PMal serves as a FRET donor, and Tyr(6) serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids. We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser(16) by PKA results in a 3 A decrease in the spatial separation between PMal at Cys(24) and nitroTyr(6) and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer(16) and Arg(13) that promotes a coil-to-helix transition. This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 A above the membrane surface. Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB. Thus, stabilization of the structure of PLB following phosphorylation of Ser(16) is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition.