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A "Warm Formamide" Scenario for the Origins of Life Might not be so Hot: Comment on "Formamide and the Origin of Life"

In this review, Saladino et al. present an intriguing hypothesis surrounding the role of formamide in the origins of life on Earth, backed by experimental results supporting each step from formamide to RNA polymers. The overall premise is that, from formamide and inorganic phosphate, RNA molecules over 100 nucleotides in length can be produced. In addition, many carboxylic acids likely relevant to prebiotic metabolism, are formed along the way. Thus, from a rather simple organic molecule that has been observed in outer space (formamide), you can generate many of the compounds necessary for the origins of life. However, because high temperatures (160 C) are required for the formamide reactions, it remains unclear where the "warm formamide" scenario could have occurred. Low-temperature, aqueous hydrogen cyanide-based prebiotic chemistry that we know actually happened has been shown to produce many of the molecules invoked in the formamide hypothesis: amino acids, carboxylic acids, sugar acids, and nucleobases have all been found in meteorites recovered on Earth, providing a plausible route for their synthesis and delivery. In contrast, a large portion of the formamide hypothesis is based on relatively hightemperature reactions, A plausible milieu for high-temperature reactions with concentrated formamide is yet to be described, and is critical for this hypothesis to be validated. Hydrothermal vents are attractive heat sources, and the higher boiling point of formamide has been invoked as a mechanism to concentrate it from an aqueous solution, Unless the water can actually evaporate, however, there would be no net enrichment. For example, in the context of a deep-sea vent, any water "removed" by heating would be quickly replaced. Some of the individual reactions underpinning the present hypothesis have been met with skepticism because they go against conventional wisdom, To name a few of the surprising results: the observation that nucleosides can be converted to cyclic phosphates when heated in the presence of minerals and inorganie phosphate; that 3'-5' cGMP and cAMP nucleotides polymerize rapidly into RNA oligomers, even in the absence of monovalent counterions and that end-to-end ligation reactions between RNA oligomers occur in essentially pure water, without requiring any activating groups or counterions. Because the polymerization reactions are simply transesterification reactions, that they readily occur in the absence of cations makes one wonder why nearly all ribozyme-catalyzed transesterification reactions are metal-ion dependent; similarly, that the end-to-end ligation reactions do not require activation runs counter to the observation that all protein-catalyzed ligation and polymerization reactions of RNA and DNA require activated substrates. Detailed mechanistic studies of the reported reactions are warranted and could provide important insights for understanding the chemistry behind the origins of life. Because the authors have produced many of the experimental results supporting their hypothesis, they could demonstrate the validity of their hypothesis by converting formamide into approx 100 nucleotide RNA oligomers, using the products of one reaction as the reactants for the next reaction, under specific conditions plausible on the pre-biotic Earth. Such a demonstration would represent a milestone for our understanding of the origins of life.

Burton, Aaron S.↗

A 'Warm Formamide' Scenario for the Origins of Life Might Not Be so Hot Comment on 'Formamide and the Origin of Life' by E. Di Mauro Et Al.

In this review, Saladino et al. present an intriguing hypothesis surrounding the role of formamide in the originsof life on Earth, backed by experimental results supporting each step from formamide to RNA polymers [1]. The overall premise is that, from formamide and inorganic phosphate, RNA molecules over 100 nucleotides in length canbe produced. In addition, many carboxylic acids likely relevant to prebiotic metabolism are formed along the way. Thus, from a rather simple organic molecule that has been observed in outer space (formamide), you can generatemany of the compounds necessary for the origins of life. However, because high temperatures (160 C) are requiredfor the formamide reactions, it remains unclear where the warm formamide scenario could have occurred.Low-temperature, aqueous hydrogen counter to the observation that all protein-catalyzed ligation and polymerization reactions of RNA and DNA requireactivated substrates. Detailed mechanistic studies of the reported reactions are warranted and could provide important insights for understanding the chemistry behind the origins of life.Because the authors have produced many of the experimental results supporting their hypothesis, they coulddemonstrate the validity of their hypothesis by converting formamide into 100 nucleotide RNA oligomers, usingthe products of one reaction as the reactants for the next reaction, under specific conditions plausible on the pre-bioticEarth. Such a demonstration would represent a milestone for our understanding of the origins of life.cyanide-based prebiotic chemistry that we know actually happened has beenshown to produce many of the molecules invoked in the formamide hypothesis: amino acids, carboxylic acids, sugaracids, and nucleobases have all been found in meteorites recovered on Earth [e.g. [24]], providing a plausible routefor their synthesis and delivery. In contrast, a large portion of the formamide hypothesis is based on relatively high-temperature reactions. A plausible milieu for high-temperature reactions with concentrated formamide is yet to bedescribed, and is critical for this hypothesis to be validated. Hydrothermal vents are attractive heat sources, and the higher boiling point of formamide has been invoked as a mechanism to concentrate it from an aqueous solution.Unless the water can actually evaporate, however, there would be no net enrichment. For example, in the context of a deep-sea vent, any water removed by heating would be quickly replaced.Some of the individual reactions underpinning the present hypothesis [1] have been met with skepticism becausethey go against conventional wisdom. To name a few of the surprising results: the observation that nucleosides can beconverted to cyclic phosphates when heated in the presence of minerals and inorganic phosphate [5]; that 35 cGMPand cAMP nucleotides polymerize rapidly into RNA oligomers, even in the absence of monovalent counterions [6];and that end-to-end ligation reactions between RNA oligomers occur in essentially pure water, without requiring any activating groups or counterions [7,8]. Because the polymerization reactions are simply transesterification reactions,that they readily occur in the absence of cations makes one wonder why nearly all ribozyme-catalyzed transesterification reactions are metal-ion dependent; similarly, that the end-to-end ligation reactions do not require activation runs.

life↗

Fine Aerosol Bulk Composition Measured on WP-3D Research Aircraft in Vicinity of the Northeastern United States - Results from NEAQS

During the New England Air Quality Study (NEAQS) in the summer of 2004, airborne measurements were made of the major inorganic ions and the water-soluble organic carbon (WSOC) of the submicron (PM(sub 1.0)) aerosol. These and ancillary data are used to describe the overall aerosol chemical characteristics encountered during the study. Fine particle mass was estimated from particle volume and a calculated density based on measured particle composition. Fine particle organic matter (OM) was estimated from WSOC and a mass balance analysis. The aerosol over the northeastern United States (U.S.) and Canada was predominantly sulfate and associated ammonium, and organic components, although in unique plumes additional ionic components were also periodically above detection limits. In power generation regions, and especially in the Ohio River Valley region, the aerosol tended to be predominantly sulfate (approximately 60% micro gram /micro gram) and apparently acidic, based on an excess of measured anions compared to cations. In all other regions where sulfate concentrations were lower and a smaller fraction of overall mass, the cations and anions were balanced suggesting a more neutral aerosol. In contrast, the WSOC and estimated OM were more spatially uniform and the fraction of OM relative to PM mass was largely influenced by sources of sulfate. The study median OM mass fraction was 40%. Throughout the study region, sulfate and organic aerosol mass were highest near the surface and decreased rapidly with increasing altitude. The relative fraction of organic mass to sulfate was similar throughout all altitudes within the boundary layer (altitude less than 2.5 km), but was significantly higher at altitude layers in the free troposphere (above 2.5 km). A number of distinct biomass burning plumes from fires in Alaska and the Yukon were periodically intercepted, mostly at altitudes between 3 and 4 km. These plumes were associated with highest aerosol concentrations of the study and were largely comprised of organic aerosol components (approximtely 60%).

Peltier, R. E.↗

Preventing Precipitation in the ISS Urine Processor

The ISS Urine Processor Assembly (UPA) was initially designed to achieve 85% recovery of water from pretreated urine on ISS. Pretreated urine is comprised of crew urine treated with flush water, an oxidant (chromium trioxide), and an inorganic acid (sulfuric acid) to control microbial growth and inhibit precipitation. Unfortunately, initial operation of the UPA on ISS resulted in the precipitation of calcium sulfate at 85% recovery. This occurred because the calcium concentration in the crew urine was elevated in microgravity due to bone loss. The higher calcium concentration precipitated with sulfate from the pretreatment acid, resulting in a failure of the UPA due to the accumulation of solids in the Distillation Assembly. Since this failure, the UPA has been limited to a reduced recovery of water from urine to prevent calcium sulfate from reaching the solubility limit. NASA personnel have worked to identify a solution that would allow the UPA to return to a nominal recovery rate of 85%. This effort has culminated with the development of a pretreatment based on phosphoric acid instead of sulfuric acid. By eliminating the sulfate associated with the pretreatment, the brine can be concentrated to a much higher concentration before calcium sulfate reach the solubility limit. This paper summarizes the development of this pretreatment and the testing performed to verify its implementation on ISS.

Muirhead, Dean↗

Waste Water Brine Purification through Electrodialysis Ion Exchange

Reutilizing resources onboard the International Space Station (ISS) and for future deep space missions are critical for mission longevity and sustainability. Waste water brine produced from water recovery systems contain chemical species that could be processed into a potential fertilizer for future plant systems.Fertilizer production can be achieved through a process called electrodialysis ion exchange. Waste water containing inorganic salt components are fed through a series of ion exchange membranes to produce fertilizer (a phosphate rich stream), electrolysis-grade water, and other useful commodities.A test bed was constructed to conduct controlled experiments and an experimental design procedure developed to determine the feasibility of the process. Conductivity and pH probes were utilized to determine the ion concentration in each of the product streams, along with ion chromatography (IC) to define the exact concentration of each ion in every stream throughout the experiment. This is crucial in order to convey the effectiveness of ion removal from the incoming waste water stream.The waste water and electrolyte streams were prepared in the lab prior to experimentation. Additionally, the ion exchange membrane configurations were developed and Opto 22 data analysis software incorporated to conduct measurements in real time.Ions successfully diffused across their respective membranes into the concentrate, acid, and base streams. This resulted in pure water, a phosphate rich stream, and a separate anion/hydrogen and cation/hydroxide stream.

Hancock, Matthew L.↗

Thermal and Evolved Gas Behavior of Calcite Under Mars Phoenix TEGA Operating Conditions

The Mars Phoenix Scout Mission with its diverse instrument suite successfully examined several soils on the Northern plains of Mars. The Thermal and Evolved Gas Analyzer (TEGA) was employed to detect organic and inorganic materials by coupling a differential scanning calorimeter (DSC) with a magnetic-sector mass spectrometer (MS). Martian soil was heated up to 1000 C in the DSC ovens and evolved gases from mineral decomposition products were examined with the MS. TEGA s DSC has the capability to detect endothermic and exothermic reactions during heating that are characteristic of minerals present in the Martian soil. Initial TEGA results indicated the presence of endothermic peaks with onset temperatures that ranged from 675 C to 750 C with corresponding CO2 release. This result suggests the presence of calcite (CaCO3. CaO + CO2). Organic combustion to CO2 is not likely since this mostly occurs at temperatures below 550 C. Fe-carbonate and Mg-carbonate are not likely because their decomposition temperatures are less than 600 C. TEGA enthalpy determinations suggest that calcite, may occur in the Martian soil in concentrations of approx.1 to 5 wt. %. The detection of calcite could be questioned based on previous results that suggest Mars soils are mostly acidic. However, the Phoenix landing site soil pH was measured at pH 8.3 0.5, which is typical of terrestrial soils where pH is controlled by calcite solubility. The range of onset temperatures and calcite concentration as calculated by TEGA is poorly con-strained in part because of limited thermal data of cal-cite at reduced pressures. TEGA operates at <30 mbar while most calcite literature thermal data was obtained at 1000 mbar or higher pressures.

Ming, D.W.↗

Mars, clays and the origins of life

To detect life in the Martian soil, tests were designed to look for respiration and photosynthesis. Both tests (labeled release, LR, and pyrolytic release, PR) for life in the Martian soils were positive. However, when the measurement for organic molecules in the soil of Mars was made, none were found. The interpretation given is that the inorganic constituents of the soil of Mars were responsible for these observations. The inorganic analysis of the soil was best fitted by a mixture of minerals: 60 to 80 percent clay, iron oxide, quartz, and soluble salts such as halite (NaCl). The minerals most successful in simulating the PR and LR experiments are iron-rich clays. There is a theory that considers clays as the first organisms capable of replication, mutation, and catalysis, and hence of evolving. Clays are formed when liquid water causes the weathering of rocks. The distribution of ions such as aluminum, magnesium, and iron play the role of bases in the DNA. The information was stored in the distribution of ions in the octahedral and tetrahedral molecules, but that they could, like RNA and DNA, replicate. When the clays replicated, each sheet of clay would be a template for a new sheet. The ion substitutions in one clay sheet would give rise to a complementary or similar pattern on the clay synthesized on its surface. It was theorized that it was on the surface of replicating iron-rich clays that carbon dioxide would be fixed in the light into organic acids such as formic or oxalic acid. If Mars had liquid water during a warm period in its past, clay formation would have been abundant. These clays would have replicated and evolved until the liquid water was removed due to cooling of Mars. It is entirely possible that the Viking mission detected life on Mars, but it was clay life that awaits the return of water to continue its evolution into life based on organic molecules.

Hartman, Hyman↗

New High-Temperature Membranes Developed for Proton Exchange Membrane Fuel Cells

Fuel cells are receiving a considerable amount of attention for potential use in a variety of areas, including the automotive industry, commercial power generation, and personal electronics. Research at the NASA Glenn Research Center has focused on the development of fuel cells for use in aerospace power systems for aircraft, unmanned air vehicles, and space transportation systems. These applications require fuel cells with higher power densities and better durability than what is required for nonaerospace uses. In addition, membrane cost is a concern for any fuel cell application. The most widely used membrane materials for proton exchange membrane (PEM) fuel cells are based on sulfonated perfluorinated polyethers, typically Nafion 117, Flemion, or Aciplex. However, these polymers are costly and do not function well at temperatures above 80 C. At higher temperatures, conventional membrane materials dry out and lose their ability to conduct protons, essential for the operation of the fuel cell. Increasing the operating temperature of PEM fuel cells from 80 to 120 C would significantly increase their power densities and enhance their durability by reducing the susceptibility of the electrode catalysts to carbon monoxide poisoning. Glenn's Polymers Branch has focused on developing new, low-cost membranes that can operate at these higher temperatures. A new series of organically modified siloxane (ORMOSIL) polymers were synthesized for use as membrane materials in a high-temperature PEM fuel cell. These polymers have an organic portion that can allow protons to transport through the polymer film and a cross-linked silica network that gives the polymers dimensional stability. These flexible xerogel polymer films are thermally stable, with decomposition onset as high as 380 C. Two types of proton-conducting ORMOSIL films have been produced: (1) NASA-A, which can coordinate many highly acid inorganic salts that facilitate proton conduction and (2) NASA-B, which has been produced and which incorporates strongly acidic (proton donating) functional groups into the polymer backbone. Both of these polymer films have demonstrated significantly higher proton conductivity than Nafion at elevated temperatures and low relative humidities. An added advantage is that these polymers are very inexpensive to produce because their starting materials are commodity chemicals that are commercially available in large volumes.

Kinder, James D.↗

Spacecraft Water Impurity Monitor, a System for Water Quality Analysis on Exploration Missions Beyond Low Earth Orbit

Exploration missions beyond low-earth-orbit (LEO) will require advanced instrumentation to monitor water quality. Traveling beyond LEO means the transfer of water samples to an Earth-based laboratory for detailed analysis is not feasible. Detailed analysis of water composition during exploration is still necessary, because having the capability to determine the specific organic chemical causing a change in total organic carbon (TOC) or the specific metal or ionic species causing a change in conductivity has the potential to inform the crew health and system management decisions. On a new vehicle such as a lunar or Mars surface habitat or a Mars transit vehicle, finding “new” impurities not seen on ISS should be expected. The key is to identify the impurity so the correct action can be taken. On ISS we can measure TOC, conductivity, and other physical properties but do not have the capability for detailed analysis using vehicle instrumentation. This is acceptable because ISS can send samples down to Earth for further analysis and obtain the detailed composition. The Spacecraft Water Impurity Monitor (SWIM) will provide detailed water quality analysis for missions in which sample down mass is not available. SWIM is a system comprising organic and inorganic analysis modules. For organic chemicals, a gas chromatograph mass spectrometer (GCMS) detects and identifies organic impurities. For inorganic species, a capillary electrophoresis capacitively coupled contactless conductivity detection (CE-C4D) system as well as ion specific electrodes detect and identify metal ions and other inorganic salts / acids. The SWIM technology demonstration project has completed a System Requirements Review (SRR) to finalize detection requirements and is currently working to refine vehicle interface requirements for a future technology demonstration. The project also has begun preliminary flight design activities for the core analyzers in the instrument suite.

Water Monitoring↗

Spacecraft Water Impurity Monitor, a System for Water Quality Analysis on Exploration Missions Beyond Low Earth Orbit

Exploration missions beyond low-earth-orbit (LEO) will require advanced instrumentation to monitor water quality. Traveling beyond LEO means the transfer of water samples to an Earth-based laboratory for detailed analysis is not feasible. Detailed analysis of water composition during exploration is still necessary, because having the capability to determine the specific organic chemical causing a change in total organic carbon (TOC) or the specific metal or ionic species causing a change in conductivity has the potential to inform the crew health and system management decisions. On a new vehicle such as a lunar or Mars surface habitat or a Mars transit vehicle, finding “new” impurities not seen on ISS should be expected. The key is to identify the impurity so the correct action can be taken. On ISS we can measure TOC, conductivity, and other physical properties but do not have the capability for detailed analysis using vehicle instrumentation. This is acceptable because ISS can send samples down to Earth for further analysis and obtain the detailed composition. The Spacecraft Water Impurity Monitor (SWIM) will provide detailed water quality analysis for missions in which sample down mass is not available. SWIM is a system comprising organic and inorganic analysis modules. For organic chemicals, a gas chromatograph mass spectrometer (GCMS) detects and identifies organic impurities. For inorganic species, a capillary electrophoresis capacitively coupled contactless conductivity detection (CE-C4D) system as well as ion specific electrodes detect and identify metal ions and other inorganic salts / acids. The SWIM technology demonstration project has completed a System Requirements Review (SRR) to finalize detection requirements and is currently working to refine vehicle interface requirements for a future technology demonstration. The project also has begun preliminary flight design activities for the core analyzers in the instrument suite.

Water Monitoring↗

Materials Combustion Testing and Combustion Product Sensor Evaluations in FY12

NASA Centers continue to collaborate to characterize the chemical species and smoke particles generated by the combustion of current space-rated non-metallic materials including fluoropolymers. This paper describes the results of tests conducted February through September 2012 to identify optimal chemical markers both for augmenting particle-based fire detection methods and for monitoring the post-fire cleanup phase in human spacecraft. These studies follow up on testing conducted in August 2010 and reported at ICES 2011. The tests were conducted at the NASA White Sands Test Facility in a custom glove box designed for burning fractional gram quantities of materials under varying heating profiles. The 623 L chamber was heavily instrumented to quantify organics (gas chromatography/mass spectrometry), inorganics by water extraction followed by ion chromatography, and select species by various individual commercially-available sensors. Evaluating new technologies for measuring carbon monoxide, hydrogen cyanide, hydrogen fluoride, hydrogen chloride and other species of interest was a key objective of the test. Some of these sensors were located inside the glovebox near the fire source to avoid losses through the sampling lines; the rest were located just outside the glovebox. Instruments for smoke particle characterization included a Tapered Element Oscillating Microbalance Personal Dust Monitor (TEOM PDM) and a TSI Dust Trak DRX to measure particle mass concentration, a TSI PTrak for number concentration and a thermal precipitator for collection of particles for microscopic analysis. Materials studied included Nomex®, M22759 wire insulation, granulated circuit board, polyvinyl chloride (PVC), Polytetrafluoroethylene (PTFE), Kapton®, and mixtures of PTFE and Kapton®. Furnace temperatures ranged from 340o to 640o C, focusing on the smoldering regime. Of particular interest in these tests was confirming burn repeatability and production of acid gases with different fuel mixture compositions, as well as the dependence of aerosol concentrations on temperature.

Smoke↗

Materials Combustion Testing and Combustion Product Sensor Evaluations in FY12

NASA Centers continue to collaborate to characterize the chemical species and smoke particles generated by the combustion of current space-rated non-metallic materials including fluoropolymers. This paper describes the results of tests conducted February through September 2012 to identify optimal chemical markers both for augmenting particle-based fire detection methods and for monitoring the post-fire cleanup phase in human spacecraft. These studies follow up on testing conducted in August 2010 and reported at ICES 2011. The tests were conducted at the NASA White Sands Test Facility in a custom glove box designed for burning fractional gram quantities of materials under varying heating profiles. The 623 L chamber was heavily instrumented to quantify organics (gas chromatography/mass spectrometry), inorganics by water extraction followed by ion chromatography, and select species by various individual commercially-available sensors. Evaluating new technologies for measuring carbon monoxide, hydrogen cyanide, hydrogen fluoride, hydrogen chloride and other species of interest was a key objective of the test. Some of these sensors were located inside the glovebox near the fire source to avoid losses through the sampling lines; the rest were located just outside the glovebox. Instruments for smoke particle characterization included a Tapered Element Oscillating Microbalance Personal Dust Monitor (TEOM PDM) and a TSI Dust Trak DRX to measure particle mass concentration, a TSI PTrak for number concentration and a thermal precipitator for collection of particles for microscopic analysis. Materials studied included Nomex(R), M22759 wire insulation, granulated circuit board, polyvinyl chloride (PVC), Polytetrafluoroethylene (PTFE), Kapton(R), and mixtures of PTFE and Kapton(R). Furnace temperatures ranged from 340 to 640 C, focusing on the smoldering regime. Of particular interest in these tests was confirming burn repeatability and production of acid gases with different fuel mixture compositions, as well as the dependence of aerosol concentrations on temperature.

Combustion↗

Synthesis of phthalocyanine doped sol-gel materials

The synthesis of sol-gel silica materials doped with three different types of metallophthalocyanines has been studied. Homogeneous materials of good optical quality were prepared and the first optical limiting measurements of dyes in sol-gel hosts were carried out. The properties of these solid state limiters are similar to limiters based on phthalocyanine (Pc) in solution. Sol-gel silica materials containing copper, tin and germanium phthalocyanines were investigated. The initial step in all cases was to prepare silica sols by the sonogel method using tetramethoxy silane (TMOS), HCl and distilled water. Thereafter, the synthesis depended upon the specific Pc and its solubility characteristics. Copper phthalocyanine tetrasulfonic acid tetra sodium salt (CuPc4S) is soluble in water and various doping levels (1 x 10 (exp -4) M to 1 x 10 (exp -5) M) were added to the sol. The group IV Pc's, SnPc(OSi(n-hexyl)3)2 and GePc(OSi(n-hexyl)3)2, are insoluble in water and the process was changed accordingly. In these cases, the compounds were dissolved in THF and then added to the sol. The Pc concentration in the sol was 2 x 10(exp -5)M. The samples were then aged and dried in the standard method of making xerogel monoliths. Comparative nanosecond optical limiting experiments were performed on silica xerogels that were doped with the different metallophthalocyanines. The ratio of the net excited state absorption cross section (sigma(sub e)) to the ground state cross section (sigma(sub g)) is an important figure of merit that is used to characterize these materials. By this standard the SnPc sample exhibits the best limiting for the Pc doped sol-gel materials. Its cross section ratio of 19 compares favorably with the value of 22 that was measured in toluene. The GePc materials appear to not be as useful as those containing SnPc. The GePc doped solids exhibit a higher onset energy (2.5 mj and lower cross section ratio, 7. The CuPc4S sol-gel material has a still lower cross section ratio, 4, however, the tetrasulfonate groups make the dye soluble in water which greatly facilitates its incorporation into the sol-gel matrix. The nonlinear transmission of CuPc4S in a pH 2 buffer solution and in a silica xerogel were compared. It is evident that the CuPc4S preserves its optical limiting behavior in the sol-gel matrix, indicating that the fundamental excited state absorption process is essentially the same for a molecule in solution or in the solid state. Although the spectroscopic details of energy level lifetimes are unknown, the significance is that passive optical limiting has been achieved in the solid state via incorporation of a dye into an inorganic host. The only compromise occurs at the extremely high energy regime where photobleaching is observed. This is a result of the limited mobility of the dye molecules in the solid silica host relative to a liquid host. The effects of photodegradation in the xerogel are additive, whereas the solution provides a supply of fresh molecules that are free to enter the active volume between pulses.

Dunn, Bruce↗

Phosphate binding protein as the biorecognition element in a biosensor for phosphate

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins. c2003 Elsevier B.V. All rights reserved.

NASA Program Fundamental Space Biology↗