Propuestas regulatorias para el diseño de un mercado ibérico a plazo eficiente de la energía = Regulatory proposals for the development of an efficient iberian energy forward market

El mercado ibérico de futuros de energía eléctrica gestionado por OMIP (“Operador do Mercado Ibérico de Energia, Pólo Português”, con sede en Lisboa), también conocido como el mercado ibérico de derivados de energía, comenzó a funcionar el 3 de julio de 2006. Se analiza la eficiencia de este mercado organizado, por lo que se estudia la precisión con la que sus precios de futuros predicen el precio de contado. En dicho mercado coexisten dos modos de negociación: el mercado continuo (modo por defecto) y la contratación mediante subasta. En la negociación en continuo, las órdenes anónimas de compra y de venta interactúan de manera inmediata e individual con órdenes contrarias, dando lugar a operaciones con un número indeterminado de precios para cada contrato. En la negociación a través de subasta, un precio único de equilibrio maximiza el volumen negociado, liquidándose todas las operaciones a ese precio. Adicionalmente, los miembros negociadores de OMIP pueden liquidar operaciones “Over-The-Counter” (OTC) a través de la cámara de compensación de OMIP (OMIClear). Las cinco mayores empresas españolas de distribución de energía eléctrica tenían la obligación de comprar electricidad hasta julio de 2009 en subastas en OMIP, para cubrir parte de sus suministros regulados. De igual manera, el suministrador de último recurso portugués mantuvo tal obligación hasta julio de 2010. Los precios de equilibrio de esas subastas no han resultado óptimos a efectos retributivos de tales suministros regulados dado que dichos precios tienden a situarse ligeramente sesgados al alza. La prima de riesgo ex-post, definida como la diferencia entre los precios a plazo y de contado en el periodo de entrega, se emplea para medir su eficiencia de precio. El mercado de contado, gestionado por OMIE (“Operador de Mercado Ibérico de la Energía”, conocido tradicionalmente como “OMEL”), tiene su sede en Madrid. Durante los dos primeros años del mercado de futuros, la prima de riesgo media tiende a resultar positiva, al igual que en otros mercados europeos de energía eléctrica y gas natural. En ese periodo, la prima de riesgo ex-post tiende a ser negativa en los mercados de petróleo y carbón. Los mercados de energía tienden a mostrar niveles limitados de eficiencia de mercado. La eficiencia de precio del mercado de futuros aumenta con el desarrollo de otros mecanismos coexistentes dentro del mercado ibérico de electricidad (conocido como “MIBEL”) –es decir, el mercado dominante OTC, las subastas de centrales virtuales de generación conocidas en España como Emisiones Primarias de Energía, y las subastas para cubrir parte de los suministros de último recurso conocidas en España como subastas CESUR– y con una mayor integración de los mercados regionales europeos de energía eléctrica. Se construye un modelo de regresión para analizar la evolución de los volúmenes negociados en el mercado continuo durante sus cuatro primeros años como una función de doce indicadores potenciales de liquidez. Los únicos indicadores significativos son los volúmenes negociados en las subastas obligatorias gestionadas por OMIP, los volúmenes negociados en el mercado OTC y los volúmenes OTC compensados por OMIClear. El número de creadores de mercado, la incorporación de agentes financieros y compañías de generación pertenecientes a grupos integrados con suministradores de último recurso, y los volúmenes OTC compensados por OMIClear muestran una fuerte correlación con los volúmenes negociados en el mercado continuo. La liquidez de OMIP está aún lejos de los niveles alcanzados por los mercados europeos más maduros (localizados en los países nórdicos (Nasdaq OMX Commodities) y Alemania (EEX)). El operador de mercado y su cámara de compensación podrían desarrollar acciones eficientes de marketing para atraer nuevos agentes activos en el mercado de contado (p.ej. industrias consumidoras intensivas de energía, suministradores, pequeños productores, compañías energéticas internacionales y empresas de energías renovables) y agentes financieros, captar volúmenes del opaco OTC, y mejorar el funcionamiento de los productos existentes aún no líquidos. Resultaría de gran utilidad para tales acciones un diálogo activo con todos los agentes (participantes en el mercado, operador de mercado de contado, y autoridades supervisoras). Durante sus primeros cinco años y medio, el mercado continuo presenta un crecimento de liquidez estable. Se mide el desempeño de sus funciones de cobertura mediante la ratio de posición neta obtenida al dividir la posición abierta final de un contrato de derivados mensual entre su volumen acumulado en la cámara de compensación. Los futuros carga base muestran la ratio más baja debido a su buena liquidez. Los futuros carga punta muestran una mayor ratio al producirse su menor liquidez a través de contadas subastas fijadas por regulación portuguesa. Las permutas carga base liquidadas en la cámara de compensación ubicada en Madrid –MEFF Power, activa desde el 21 de marzo de 2011– muestran inicialmente valores altos debido a bajos volúmenes registrados, dado que esta cámara se emplea principalmente para vencimientos pequeños (diario y semanal). Dicha ratio puede ser una poderosa herramienta de supervisión para los reguladores energéticos cuando accedan a todas las transacciones de derivados en virtud del Reglamento Europeo sobre Integridad y Transparencia de los Mercados de Energía (“REMIT”), en vigor desde el 28 de diciembre de 2011. La prima de riesgo ex-post tiende a ser positiva en todos los mecanismos (futuros en OMIP, mercado OTC y subastas CESUR) y disminuye debido a la curvas de aprendizaje y al efecto, desde el año 2011, del precio fijo para la retribución de la generación con carbón autóctono. Se realiza una comparativa con los costes a plazo de generación con gas natural (diferencial “clean spark spread”) obtenido como la diferencia entre el precio del futuro eléctrico y el coste a plazo de generación con ciclo combinado internalizando los costes de emisión de CO2. Los futuros eléctricos tienen una elevada correlación con los precios de gas europeos. Los diferenciales de contratos con vencimiento inmediato tienden a ser positivos. Los mayores diferenciales se dan para los contratos mensuales, seguidos de los trimestrales y anuales. Los generadores eléctricos con gas pueden maximizar beneficios con contratos de menor vencimiento. Los informes de monitorización por el operador de mercado que proporcionan transparencia post-operacional, el acceso a datos OTC por el regulador energético, y la valoración del riesgo regulatorio pueden contribuir a ganancias de eficiencia. Estas recomendaciones son también válidas para un potencial mercado ibérico de futuros de gas, una vez que el hub ibérico de gas –actualmente en fase de diseño, con reuniones mensuales de los agentes desde enero de 2013 en el grupo de trabajo liderado por el regulador energético español– esté operativo. El hub ibérico de gas proporcionará transparencia al atraer más agentes y mejorar la competencia, incrementando su eficiencia, dado que en el mercado OTC actual no se revela precio alguno de gas. ABSTRACT The Iberian Power Futures Market, managed by OMIP (“Operador do Mercado Ibérico de Energia, Pólo Português”, located in Lisbon), also known as the Iberian Energy Derivatives Market, started operations on 3 July 2006. The market efficiency, regarding how well the future price predicts the spot price, is analysed for this energy derivatives exchange. There are two trading modes coexisting within OMIP: the continuous market (default mode) and the call auction. In the continuous trading, anonymous buy and sell orders interact immediately and individually with opposite side orders, generating trades with an undetermined number of prices for each contract. In the call auction trading, a single price auction maximizes the traded volume, being all trades settled at the same price (equilibrium price). Additionally, OMIP trading members may settle Over-the-Counter (OTC) trades through OMIP clearing house (OMIClear). The five largest Spanish distribution companies have been obliged to purchase in auctions managed by OMIP until July 2009, in order to partly cover their portfolios of end users’ regulated supplies. Likewise, the Portuguese last resort supplier kept that obligation until July 2010. The auction equilibrium prices are not optimal for remuneration purposes of regulated supplies as such prices seem to be slightly upward biased. The ex-post forward risk premium, defined as the difference between the forward and spot prices in the delivery period, is used to measure its price efficiency. The spot market, managed by OMIE (Market Operator of the Iberian Energy Market, Spanish Pool, known traditionally as “OMEL”), is located in Madrid. During the first two years of the futures market, the average forward risk premium tends to be positive, as it occurs with other European power and natural gas markets. In that period, the ex-post forward risk premium tends to be negative in oil and coal markets. Energy markets tend to show limited levels of market efficiency. The price efficiency of the Iberian Power Futures Market improves with the market development of all the coexistent forward contracting mechanisms within the Iberian Electricity Market (known as “MIBEL”) – namely, the dominant OTC market, the Virtual Power Plant Auctions known in Spain as Energy Primary Emissions, and the auctions catering for part of the last resort supplies known in Spain as CESUR auctions – and with further integration of European Regional Electricity Markets. A regression model tracking the evolution of the traded volumes in the continuous market during its first four years is built as a function of twelve potential liquidity drivers. The only significant drivers are the traded volumes in OMIP compulsory auctions, the traded volumes in the OTC market, and the OTC cleared volumes by OMIClear. The amount of market makers, the enrolment of financial members and generation companies belonging to the integrated group of last resort suppliers, and the OTC cleared volume by OMIClear show strong correlation with the traded volumes in the continuous market. OMIP liquidity is still far from the levels reached by the most mature European markets (located in the Nordic countries (Nasdaq OMX Commodities) and Germany (EEX)). The market operator and its clearing house could develop efficient marketing actions to attract new entrants active in the spot market (e.g. energy intensive industries, suppliers, small producers, international energy companies and renewable generation companies) and financial agents as well as volumes from the opaque OTC market, and to improve the performance of existing illiquid products. An active dialogue with all the stakeholders (market participants, spot market operator, and supervisory authorities) will help to implement such actions. During its firs five and a half years, the continuous market shows steady liquidity growth. The hedging performance is measured through a net position ratio obtained from the final open interest of a month derivatives contract divided by its accumulated cleared volume. The base load futures in the Iberian energy derivatives exchange show the lowest ratios due to good liquidity. The peak futures show bigger ratios as their reduced liquidity is produced by auctions fixed by Portuguese regulation. The base load swaps settled in the clearing house located in Spain – MEFF Power, operating since 21 March 2011, with a new denomination (BME Clearing) since 9 September 2013 – show initially large values due to low registered volumes, as this clearing house is mainly used for short maturity (daily and weekly swaps). The net position ratio can be a powerful oversight tool for energy regulators when accessing to all the derivatives transactions as envisaged by European regulation on Energy Market Integrity and Transparency (“REMIT”), in force since 28 December 2011. The ex-post forward risk premium tends to be positive in all existing mechanisms (OMIP futures, OTC market and CESUR auctions) and diminishes due to the learning curve and the effect – since year 2011 – of the fixed price retributing the indigenous coal fired generation. Comparison with the forward generation costs from natural gas (“clean spark spread”) – obtained as the difference between the power futures price and the forward generation cost with a gas fired combined cycle plant taking into account the CO2 emission rates – is also performed. The power futures are strongly correlated with European gas prices. The clean spark spreads built with prompt contracts tend to be positive. The biggest clean spark spreads are for the month contract, followed by the quarter contract and then by the year contract. Therefore, gas fired generation companies can maximize profits trading with contracts of shorter maturity. Market monitoring reports by the market operator providing post-trade transparency, OTC data access by the energy regulator, and assessment of the regulatory risk can contribute to efficiency gains. The same recommendations are also valid for a potential Iberian gas futures market, once an Iberian gas hub – currently in a design phase, with monthly meetings amongst the stakeholders in a Working Group led by the Spanish energy regulatory authority since January 2013 – is operating. The Iberian gas hub would bring transparency attracting more shippers and improving competition and thus its efficiency, as no gas price is currently disclosed in the existing OTC market.

Self-consistent relativistic band structure of the CH3NH3PbI3 perovskite

The electronic structure and properties of the orthorhombic phase of the CH 3 NH 3 PbI 3 perovskite are computed with density functional theory. The structure, optimized using a van der Waals functional, reproduces closely the unit cell volume. The experimental band gap is reproduced accurately by combining spin-orbit effects and a hybrid functional in which the fraction of exact exchange is tuned self-consistently to the optical dielectric constant. Including spin-orbit coupling strongly reduces the anisotropy of the effective mass tensor, predicting a low electron effective mass in all crystal directions. The computed binding energy of the unrelaxed exciton agrees with experimental data, and the values found imply a fast exciton dissociation at ambient temperature. Also polaron masses for the separated carriers are estimated. The values of all these parameters agree with recent indications that fast dynamics and large carrier diffusion lengths are key in the high photovoltaic efficiencies shown by these materials.

Functional response of Ulmus minor Mill. to drought, flooding and dutch elm disease

Ulmus minor es una especie arbórea originaria de Europa cuyas poblaciones han sido diezmadas por el hongo patógeno causante de la enfermedad de la grafiosis. La conservación de los olmos exige plantearse su propagación a través de plantaciones y conocer mejor su ecología y biología. Ulmus minor es un árbol de ribera, pero frecuentemente se encuentra alejado del cauce de arroyos y ríos, donde la capa freática sufre fuertes oscilaciones. Por ello, nuestra hipótesis general es que esta especie es moderadamente resistente tanto a la inundación como a la sequía. El principal objetivo de esta tesis doctoral es entender desde un punto de vista funcional la respuesta de U. minor a la inundación, la sequía y la infección por O. novo-ulmi; los factores que posiblemente más influyen en la distribución actual de U. minor. Con este objetivo se persigue dar continuidad a los esfuerzos de conservación de esta especie que desde hace años se dedican en varios centros de investigación a nivel mundial, ya que, entender mejor los mecanismos que contribuyen a la resistencia de U. minor ante la inoculación con O. novo-ulmi y factores de estrés abiótico ayudará en la selección y propagación de genotipos resistentes a la grafiosis. Se han planteado tres experimentos en este sentido. Primero, se ha comparado la tolerancia de brinzales de U. minor y U. laevis – otro olmo ibérico – a una inmersión controlada con el fin de evaluar su tolerancia a la inundación y comprender los mecanismos de aclimatación. Segundo, se ha comparado la tolerancia de brinzales de U. minor y Quercus ilex – una especie típica de ambientes Mediterránea secos – a la falta de agua en el suelo con el fin de evaluar el grado de tolerancia y los mecanismos de aclimatación a la sequía. El hecho de comparar dos especies contrastadas responde al interés en entender mejor cuales son los procesos que conducen a la muerte de una planta en condiciones de sequía – asunto sobre el que hay una interesante discusión desde hace algunos años. En tercer lugar, con el fin de entender mejor la resistencia de algunos genotipos de U. minor a la grafiosis, se han estudiado las diferencias fisiológicas y químicas constitutivas e inducidas por O. novo-ulmi entre clones de U. minor seleccionados a priori por su variable grado de resistencia a esta enfermedad. En el primer experimento se observó que los brinzales de U. minor sobrevivieron 60 días inmersos en una piscina con agua no estancada hasta una altura de 2-3 cm por encima del cuello de la raíz. A los 60 días, los brinzales de U. laevis se sacaron de la piscina y, a lo largo de las siguientes semanas, fueron capaces de recuperar las funciones fisiológicas que habían sido alteradas anteriormente. La conductividad hidráulica de las raíces y la tasa de asimilación de CO2 neta disminuyeron en ambas especies. Por el contrario, la tasa de respiración de hojas, tallos y raíces aumentó en las primeras semanas de la inundación, posiblemente en relación al aumento de energía necesario para desarrollar mecanismos de aclimatación a la inundación, como la hipertrofia de las lenticelas que se observó en ambas especies. Por ello, el desequilibrio del balance de carbono de la planta podría ser un factor relevante en la mortalidad de las plantas ante inundaciones prolongadas. Las plantas de U. minor (cultivadas en envases de 16 litros a media sombra) sobrevivieron por un prolongado periodo de tiempo en verano sin riego; la mitad de las plantas murieron tras 90 días sin riego. El cierre de los estomas y la pérdida de hojas contribuyeron a ralentizar las pérdidas de agua y tolerar la sequía en U. minor. Las obvias diferencias en tolerancia a la sequía con respecto a Q. ilex se reflejaron en la distinta capacidad para ralentizar la aparición del estrés hídrico tras dejar de regar y para transportar agua en condiciones de elevada tensión en el xilema. Más relevante es que las plantas con evidentes síntomas de decaimiento previo a su muerte exhibieron pérdidas de conductividad hidráulica en las raíces del 80% en ambas especies, mientras que las reservas de carbohidratos apenas variaron y lo hicieron de forma desigual en ambas especies. Árboles de U. minor de 5 y 6 años de edad (plantados en eras con riego mantenido) exhibieron una respuesta a la inoculación con O. novo-ulmi consistente con ensayos previos de resistencia. La conductividad hidráulica del tallo, el potencial hídrico foliar y la tasa de asimilación de CO2 neta disminuyeron significativamente en relación a árboles inoculados con agua, pero solo en los clones susceptibles. Este hecho enlaza con el perfil químico “más defensivo” de los clones resistentes, es decir, con los mayores niveles de suberina, ácidos grasos y compuestos fenólicos en estos clones que en los susceptibles. Ello podría restringir la propagación del hongo en el árbol y preservar el comportamiento fisiológico de los clones resistentes al inocularlos con el patógeno. Los datos indican una respuesta fisiológica común de U. minor a la inundación, la sequía y la infección por O. novo-ulmi: pérdida de conductividad hidráulica, estrés hídrico y pérdida de ganancia neta de carbono. Pese a ello, U. minor desarrolla varios mecanismos que le confieren una capacidad moderada para vivir en suelos temporalmente anegados o secos. Por otro lado, el perfil químico es un factor relevante en la resistencia de ciertos genotipos a la grafiosis. Futuros estudios deberían examinar como este perfil químico y la resistencia a la grafiosis se ven alteradas por el estrés abiótico. ABSTRACT Ulmus minor is a native European elm species whose populations have been decimated by the Dutch elm disease (DED). An active conservation of this species requires large-scale plantations and a better understanding of its biology and ecology. U. minor generally grows close to water channels. However, of the Iberian riparian tree species, U. minor is the one that spread farther away from rivers and streams. For these reasons, we hypothesize that this species is moderately tolerant to both flooding and drought stresses. The main aim of the present PhD thesis is to better understand the functional response of U. minor to the abiotic stresses – flooding and drought – and the biotic stress – DED – that can be most influential on its distribution. The overarching goal is to aid in the conservation of this emblematic species through a better understanding of the mechanisms that contribute to resistance to abiotic and biotic stresses; an information that can help in the selection of resistant genotypes and their expansion in large-scale plantations. To this end, three experiments were set up. First, we compared the tolerance to experimental immersion between seedlings of U. minor and U. laevis – another European riparian elm species – in order to assess their degree of tolerance and understand the mechanisms of acclimation to this stress. Second, we investigated the tolerance to drought of U. minor seedlings in comparison with Quercus ilex (an oak species typical of dry Mediterranean habitats). Besides assessing and understanding U. minor tolerance to drought at the seedling stage, the aim was to shed light into the functional alterations that trigger drought-induced plant mortality – a matter of controversy in the last years. Third, we studied constitutive and induced physiological and biochemical differences among clones of variable DED resistance, before and following inoculation with Ophiostoma novo-ulmi. The goal is to shed light into the factors of DED resistance that is evident in some genotypes of U. minor, but not others. Potted seedlings of U. minor survived for 60 days immersed in a pool with running water to approximately 2-3 cm above the stem collar. By this time, U. minor seedlings died, whereas U. laevis seedlings moved out of the pool were able to recover most physiological functions that had been altered by flooding. For example, root hydraulic conductivity and leaf photosynthetic CO2 uptake decreased in both species; while respiration initially increased with flooding in leaves, stems and roots possibly to respond to energy demands associated to mechanisms of acclimation to soil oxygen deficiency; as example, a remarkable hypertrophy of lenticels was soon observed in flooded seedlings of both species. Therefore, the inability to maintain a positive carbon balance somehow compromises seedling survival under flooding, earlier in U. minor than U. laevis, partly explaining their differential habitats. Potted seedlings of U. minor survived for a remarkable long time without irrigation – half of plants dying only after 90 days of no irrigation in conditions of high vapour pressure deficit typical of summer. Some mechanisms that contributed to tolerate drought were leaf shedding and stomata closure, which reduced water loss and the risk of xylem cavitation. Obviously, U. minor was less tolerant to drought than Q. ilex, differences in drought tolerance resulting mostly from the distinct capacity to postpone water stress and conduct water under high xylem tension among species. More relevant was that plants of both species exhibited similar symptoms of root hydraulic failure (i.e. approximately 80% loss of hydraulic conductivity), but a slight and variable depletion of non-structural carbohydrate reserves preceding dieback. Five- and six-year-old trees of U. minor (planted in the field with supplementary watering) belonging to clones of contrasted susceptibility to DED exhibited a different physiological response to inoculation with O. novo-ulmi. Stem hydraulic conductivity, leaf water potential and photosynthetic CO2 uptake decreased significantly relative to control trees inoculated with water only in DED susceptible clones. This is consistent with the “more defensive” chemical profile observed in resistant clones, i.e. with higher levels of saturated hydrocarbons (suberin and fatty acids) and phenolic compounds than in susceptible clones. These compounds could restrict the spread of O. novo-ulmi and contribute to preserving the near-normal physiological function of resistant trees when exposed to the pathogen. These results evidence common physiological responses of U. minor to flooding, drought and pathogen infection leading to xylem water disruption, leaf water stress and reduced net carbon gain. Still, seedlings of U. minor develop various mechanisms of acclimation to abiotic stresses that can play a role in surviving moderate periods of flood and drought. The chemical profile appears to be an important factor for the resistance of some genotypes of U. minor to DED. How abiotic stresses such as flooding and drought affect the capacity of resistant U. minor clones to face O. novo-ulmi is a key question that must be contemplated in future research.

Low-Intensity Pulsed Ultrasound Improves the Functional Properties of Cardiac Mesoangioblasts

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a noninvasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5´-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of β1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Efficient exchange of the primary quinone acceptor QA in isolated reaction centers of Rhodopseudomonas viridis

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, QA and QB, where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura et al. [Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975) Proc. Natl. Acad. Sci. USA 88, 3491–3495], who were able to extract with detergents the firmly bound ubiquinone QA from the RC of Rhodobacter sphaeroides and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of Rhodopseudomonas viridis despite the fact that its QA site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of QA with the RC protein. We establish that a simple incubation procedure of isolated RCs of Rp. viridis with an excess of extraneous quinone allows the menaquinone-9 in the QA site to be almost quantitatively replaced either by vitamin K1, a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K1 with the protein at the QA site of Rp. viridis and Rb. sphaeroides, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone–protein interactions by comparison with the crystallographic data available for the QA site of the two RCs.

Evolution of the folding ability of proteins through functional selection

An evolutionary process is simulated with a simple spin-glass-like model of proteins to examine the origin of folding ability. At each generation, sequences are randomly mutated and subjected to a simulation of the folding process based on the model. According to the frequency of local configurations at the active sites, sequences are selected and passed to the next generation. After a few hundred generations, a sequence capable of folding globally into a native conformation emerges. Moreover, the selected sequence has a distinct energy minimum and an anisotropic funnel on the energy surface, which are the imperative features for fast folding of proteins. The proposed model reveals that the functional selection on the local configurations leads a sequence to fold globally into a conformation at a faster rate.

RecA tests homology at both pairing and strand exchange

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2–14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.

KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

ATP-sensitive potassium (“KATP”) channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The KATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (Kir6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic β cell KATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the Kir6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na+- and K+-dependent pH homeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H+, Na+/H+, and K+/H+ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H+ and Na+/H+ antiports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 β-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

Cerebral energetics and the glycogen shunt: Neurochemical basis of functional imaging

Positron-emission tomography and functional MRS imaging signals can be analyzed to derive neurophysiological values of cerebral blood flow or volume and cerebral metabolic consumption rates of glucose (CMRGlc) or oxygen (CMRO2). Under basal physiological conditions in the adult mammalian brain, glucose oxidation is nearly complete so that the oxygen-to-glucose index (OGI), given by the ratio of CMRO2/CMRGlc, is close to the stoichiometric value of 6. However, a survey of functional imaging data suggests that the OGI is activity dependent, moving further below the oxidative value of 6 as activity is increased. Brain lactate concentrations also increase with stimulation. These results had led to the concept that brain activation is supported by anaerobic glucose metabolism, which was inconsistent with basal glucose oxidation. These differences are resolved here by a proposed model of glucose energetics, in which a fraction of glucose is cycled through the cerebral glycogen pool, a fraction that increases with degree of brain activation. The “glycogen shunt,” although energetically less efficient than glycolysis, is followed because of its ability to supply glial energy in milliseconds for rapid neurotransmitter clearance, as a consequence of which OGI is lowered and lactate is increased. The value of OGI observed is consistent with passive lactate efflux, driven by the observed lactate concentration, for the few experiments with complete data. Although the OGI changes during activation, the energies required per neurotransmitter release (neuronal) and clearance (glial) are constant over a wide range of brain activity.

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable11

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) by its Ca2+-insensitive protein-serine/threonine kinase (PEPC kinase) in the C4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m−2 s−1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO2-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.

Photochemical energy conversion in a helical oligoproline assembly.

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.