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NASA NTRS1999

A liposomal model that mimics the cutaneous production of vitamin D3. Studies of the mechanism of the membrane-enhanced thermal isomerization of previtamin D3 to vitamin D3

We reported previously that the rate of previtamin D3 (preD3) <==> vitamin D3 isomerization was enhanced by about 10 times in the skin compared with that in organic solvents. To elucidate the mechanism by which the rate of this reaction is enhanced in the skin, we developed a liposomal model that mimicked the enhanced isomerization of preD3 to vitamin D3 that was described in human skin. Using this model we studied the effect of changing the polarity of preD3 as well as changing the chain length and the degree of saturation of liposomal phospholipids on the kinetics of preD3 <==> vitamin D3 isomerization. We found that a decrease in the hydrophilic interaction of the preD3 with liposomal phospholipids by an esterification of the 3beta-hydroxy of preD3 (previtamin D3-3beta-acetate) reduced the rate of the isomerization by 67%. The addition of a hydroxyl on C-25 of the hydrophobic side chain (25-hydroxyprevitamin D3), which decreased the hydrophobic interaction of preD3 with the phospholipids, reduced the rate by 87%. In contrast, in an isotropic n-hexane solution, there was little difference among the rates of the conversion of preD3, its 3beta-acetate, and 25-hydroxy derivatives to their corresponding vitamin D3 compounds. We also determined rate constants (k) of preD3 <==> vitamin D3 isomerization in liposomes containing phosphatidylcholines with different carbon chain lengths. The rates of the reaction were found to be enhanced as the number of carbons (Cn) in the hydrocarbon chain of the phospholipids increased from 10 to 18. In conclusion, these results support our hypothesis that amphipathic interactions between preD3 and membrane phospholipids stabilize preD3 in its "cholesterol like" cZc-conformer, the only conformer of preD3 that can convert to vitamin D3. The stronger these interactions were, the more preD3 was likely in its cZc conformation at any moment and the faster was the rate of its conversion to vitamin D3.

NASA Discipline Regulatory Physiology↗

NASA NTRS2004

Curved-Focal-Plane Arrays Using Deformed-Membrane Photodetectors

A versatile and simple approach to the design and fabrication of curved-focal-plane arrays of silicon-based photodetectors is being developed. This approach is an alternative to the one described in "Curved Focal-Plane Arrays Using Back- Illuminated High-Purity Photodetectors" (NPO-30566), NASA Tech Briefs, Vol. 27, No. 10 (October 2003), page 10a. As in the cited prior article, the basic idea is to improve the performance of an imaging instrument and simplify the optics needed to obtain a given level of performance by making an image sensor (in this case, an array of photodetectors) conform to a curved focal surface, instead of designing the optics to project an image onto a flat focal surface. There is biological precedent for curved-focal-surface designs: retinas - the image sensors in eyes - conform to the naturally curved focal surfaces of eye lenses. The present approach is applicable to both front-side- and back-side-illuminated, membrane photodetector arrays and is being demonstrated on charge-coupled devices (CCDs). The very-large scale integrated (VLSI) circuitry of such a CCD or other array is fabricated on the front side of a silicon substrate, then the CCD substrate is attached temporarily to a second substrate for mechanical support, then material is removed from the back to obtain the CCD membrane, which typically has a thickness between 10 and 20 m. In the case of a CCD designed to operate in back-surface illumination, delta doping can be performed after thinning to enhance the sensitivity. This approach is independent of the design and method of fabrication of the front-side VLSI circuitry and does not involve any processing of a curved silicon substrate. In this approach, a third substrate would be prepared by polishing one of its surfaces to a required focal-surface curvature. A CCD membrane fabricated as described above would be pressed against, deformed into conformity with, and bonded to, the curved surface. The technique used to press and bond the CCD membrane would depend on the nature of the supporting material (see figure). For example, if the third substrate were made of quartz frit, the substrate would be prepared by suffusing it with epoxy. Then one would take advantage of the porosity of the frit by applying a partial vacuum to the opposite surface of the frit, causing atmospheric pressure to push the CCD membrane against the curved surface. The curing of the epoxy would bond the CCD membrane to the curved surface. Alternatively, if the third substrate were made of a nonporous material, the curved substrate surface would be prepared by coating it with a wax or an uncured epoxy. The CCD membrane would be pressed against the coated, curved surface by use of a suitably pressurized balloon. The CCD membrane would then become bonded to the curved surface by curing of the epoxy or freezing of the wax.

Nikzad, Shouleh↗

NASA NTRS2011

Technique for Configuring an Actively Cooled Thermal Shield in a Flight System

Broad area cooling shields are a mass-efficient alternative to conductively cooled thermal radiation shielding. The shield would actively intercept a large portion of incident thermal radiation and transport the heat away using cryogenic helium gas. The design concept consists of a conductive and conformable surface that maximizes heat transfer and formability. Broad Area Cooled (BAC) shields could potentially provide considerable mass savings for spaceflight applications by eliminating the need for a rigid thermal radiation shield for cryogen tanks. The BAC consists of a network of capillary tubes that are thermally connected to a conductive shield material. Chilled helium gas is circulated through the network and transports unwanted heat away from the cryogen tanks. The cryogenic helium gas is pumped and chilled simultaneously using a specialized pulse-tube cryocooler, which further improves the mass efficiency of the system. By reducing the thermal environment temperature from 300 to 100 K, the radiative heat load on a cryogen tank could be reduced by an order of magnitude. For a cryogenic liquid propellant scenario of oxygen and hydrogen, the boiloff of hydrogen would be significantly reduced and completely eliminated for oxygen. A major challenge in implementing this technology on large tanks is that the BAC system must be easily scalable from lab demonstrations to full-scale missions. Also, the BAC shield must be conformable to complex shapes like spheres without losing the ability to maintain constant temperature throughout. The initial design maximizes thermal conductivity between the capillary tube and the conductive radiation shielding by using thin, corrugated aluminum foil with the tube running transverse to the folds. This configuration has the added benefit of enabling the foil to stretch and contract longitudinally. This allows the BAC to conform to the complex curvature of a cryogen tank, which is key to its success. To demonstrate a BAC shield system with minimal impact to current cryogen tank designs, the shielding must be applied after the final assembly of the tank and supporting structure. One method is to pre-fabricate the shield in long strips. A spool of corrugated aluminum foil with a thermally sunk aluminum capillary running through the center could then be simply wound around the cryogen tanks and encapsulated within the multi-layer insulation (MLI) blanket. Then, on orbit, the BAC would intercept thermal radiation coming in through the MLI and transport it away from the cryogen tanks. An optimization of the design could be done to take into account mass savings from thinner MLI blankets, eliminating solid thermal shields, and ultimately, a reduction in the required cryogen tank size.

Barkfknecht, Peter↗

NASA NTRS2013

Medicine Delivery Device with Integrated Sterilization and Detection

Sheam, Michael J.↗

NASA NTRS2008

Measurement Assurance for End-Item Users

The goal of a Quality Management System (QMS) as specified in ISO 9001 and AS9100 is to assure the end product meets specifications and customer requirements. Measuring devices, often called measuring and test equipments (MTE), provide the evidence of product conformity to the prescribed requirements. Therefore the processes which employ MTE can become a weak link to the overall QMS if proper attention is not given to development and execution of these processes. Traditionally, calibration of MTE is given more focus in industry standards and process control efforts than the equally important proper usage of the same equipment. It is a common complaint of calibration laboratory personnel that MTE users are only interested in "a sticker." If the QMS requires the MTE "to demonstrate conformity of the product," then the quality of the measurement process must be adequate for the task. This leads to an ad hoc definition; measurement assurance is a discipline that assures that all processes, activities, environments, standards, and procedures involved in making a measurement produce a result that can be rigorously evaluated for validity and accuracy. To evaluate that the existing measurement processes are providing an adequate level of quality to support the decisions based upon this measurement data, an understanding of measurement assurance basics is essential. This topic is complimentary to the calibration standard, ANSI/NCSL Z540.3-2006, which targets the calibration of MTE at the organizational level. This paper will discuss general measurement assurance when MTE is used to provide evidence of product conformity, therefore the target audience of this paper is end item users of MTE. A central focus of the paper will be the verification of tolerances and the associated risks, so calibration professionals may find the paper useful in communication with their customers, MTE users.

Mimbs, Scott M.↗

NASA NTRS2013

Medicine Delivery Device with Integrated Sterilization and Detection

Sterile delivery devices can be created by integrating a medicine delivery instrument with surfaces that are coated with germicidal and anti-fouling material. This requires that a large-surface-area template be developed within a constrained volume to ensure good contact between the delivered medicine and the germicidal material. Both of these can be integrated using JPL-developed silicon nanotip or cryo-etch black silicon technologies with atomic layer deposition (ALD) coating of specific germicidal layers. The application of semiconductor processing techniques and technologies to the problems of fluid manipulation and delivery has enabled the integration of chemical, electrical, and mechanical manipulation of samples all within a single microfluidic device. This approach has been successfully applied at JPL to the automated processing, detection, and analysis of minute quantities (parts per trillion level) of biomaterials to develop instruments for in situ exploration or extraterrestrial bodies. The same nanofabrication techniques that are used to produce a microfluidics device are also capable of synthesizing extremely high-surface-area templates in precise locations, and coating those surfaces with conformal films to manipulate their surface properties. This methodology has been successfully applied at JPL to produce patterned and coated silicon nanotips (also known as black silicon) to manipulate the hydrophilicity of surfaces to direct the spreading of fluids in microdevices. JPL's ALD technique is an ideal method to produce the highly conformal coatings required for this type of application. Certain materials, such as TiO2, have germicidal and anti-fouling properties when they are illuminated with UV light. The proposed delivery device contacts medicine with this high-surface-area black silicon surface coated with a thin-film germicidal deposited conformally with ALD. The coating can also be illuminated with ultraviolet light for the purpose of sterilization or identification of the medicine itself. This constrained volume that is located immediately prior to delivery into a patient, ensures that the medicine delivery device is inherently sterile. An additional benefit to integrating a high-surface-area template within the fluid channel of a medicine delivery device is that one can envision a number of different functional coatings that could facilitate the capture and analysis of either microbial contaminants or the medicine itself. For example, one could attach antibodies or some other binding agent with a specific affinity to the silicon nanotip template. Once a target molecule or microbe is bound to the high-surface- area template, one could use an optical analytical technique such as fluorescence or adsorption to determine the identity and potentially the concentration of the species of interest. By illuminating the bound species from the back, it may also be possible to probe only the molecules with an evanescent wave, making detection of the species from the front side of the device much simpler.

Shearn, Michael J.↗

NASA NTRS2016

Separation Assurance and Scheduling Coordination in the Arrival Environment

Separation assurance (SA) automation has been proposed as either a ground-based or airborne paradigm. The arrival environment is complex because aircraft are being sequenced and spaced to the arrival fix. This paper examines the effect of the allocation of the SA and scheduling functions on the performance of the system. Two coordination configurations between an SA and an arrival management system are tested using both ground and airborne implementations. All configurations have a conflict detection and resolution (CD&R) system and either an integrated or separated scheduler. Performance metrics are presented for the ground and airborne systems based on arrival traffic headed to Dallas/ Fort Worth International airport. The total delay, time-spacing conformance, and schedule conformance are used to measure efficiency. The goal of the analysis is to use the metrics to identify performance differences between the configurations that are based on different function allocations. A surveillance range limitation of 100 nmi and a time delay for sharing updated trajectory intent of 30 seconds were implemented for the airborne system. Overall, these results indicate that the surveillance range and the sharing of trajectories and aircraft schedules are important factors in determining the efficiency of an airborne arrival management system. These parameters are not relevant to the ground-based system as modeled for this study because it has instantaneous access to all aircraft trajectories and intent. Creating a schedule external to the CD&R and the scheduling conformance system was seen to reduce total delays for the airborne system, and had a minor effect on the ground-based system. The effect of an external scheduler on other metrics was mixed.

function allocation↗

NASA NTRS2018

Estimating a Large Phased Array Antenna Radiation Pattern by Computer Electromagnetic Simulation

The design of a conformal antenna for use on UAV's (Unmanned Aerial Vehicle) and other aircraft can be enhanced with computational electromagnetic modeling to determine the expected radiation patterns of the antenna when mounted on the aircraft. However, detailed simulation of the antenna structure and the aircraft together requires significant computational resources and time which may not be available. This paper details several methods for estimating the radiation pattern of a 50 by 50-element patch antenna. The Conformal Lightweight Antenna Structures for Aeronautical Communication Technologies (CLASACT) Program aims to build and test a 14.25 gigaherz (Ku-band) conformal antenna on a NASA-owned UAV. The antenna is intended for satellite communications and will enable communication between a ground station and a UAV when the separation distance is too great for line-of-sight communication. It is estimated that a 2 degree beamwidth will be necessary, requiring a 50 by 50 array of patch elements. The narrow beamwidth requirement, together with an element spacing of 0.6 lambda means that the array length will be 30 wavelengths, electrically very large. Three methods of varying complexity are described for estimating the total far-field radiation pattern. The results are shown for each method in the form of a normalized power pattern.

Mackenzie, Anne I.↗

NASA NTRS2018

Printed Wiring Board Technology Infusion and Supplier Capability Overview

Risk based technology infusion is a deliberate and systematic process which defines the analysis and communication methodology by which new technology is applied and integrated into existing and new designs, identifies technology development needs based on trends analysis and facilitates the identification of shortfalls against performance objectives. This presentation at QLF 2018 provides the audience a snapshot of quality variations in printed wiring board quality, as assessed, using experiences in processing and risk analysis of PWB structural integrity coupons. The presentation will focus on printed wiring board quality metrics used, the relative type and number of non-conformances observed and trend analysis using statistical methods. Trend analysis shows the top five non-conformances observed across PWB suppliers, the root cause(s) behind these non-conformance and suggestions of mitigation plans. The trends will then be matched with the current state of the PWB supplier base and its challenges and opportunities. The presentation further discusses the methods being applied at GSFC for evaluating candidate printed wiring board technologies which promote the adoption of higher throughput and faster processing technology for GSFC missions.

PCB fabrication↗

NASA NTRS2019

Swept Volume Approach for the Characterization of Pumping Loss of Shrouded Meshed Cylindrical Gears

High speed rotorcraft transmissions are subject to load-independent power losses consisting of drag and pumping loss. Tightly conforming shrouds enclosing the transmission gears are often incorporated to reduce the drag component of the total load-independent losses. However, tightly conforming axial shrouding can result in an increase in the pumping loss component. Quantifying the pumping loss of shrouded gear transmissions has been the subject of many studies. This study presents a new approach for estimating pumping loss based on the concept of swept volume borrowed from the positive displacement pump and compressor industry. In this study, pumping loss of shrouded gear transmissions is considered to be related to the swept volume of the gear sets and the downstream flow resistance created by the shroud clearances. The drag loss and pumping loss of a spur gear pair have been determined through testing using the NASA Glenn Research Center Gear Windage Test Facility. The results from this testing have been compared to theoretical results using the formulations presented in this study. Good correlation exist between the test pumping power loss and the predicted pumping power loss for tightly conforming axial shroud configurations.

Hurrell, Michael J.↗

NASA NTRS2015

Arrival Scheduling with Shortcut Path Options and Mixed Aircraft Performance

Previous work introduced the concept of using tactical shortcut options to improve schedule conformance in terminal airspace. When a scheduling point is congested, aircraft are scheduled to longer nominal paths, holding shortcut path options in reserve for tactical use if an aircraft is late, thereby improving the schedule conformance, reducing the required scheduling buffer, and increasing throughput. When the scheduling point is less congested, aircraft may be scheduled to the shorter path with original larger scheduling buffers. Previous work focused on a single generic merge point serving aircraft with uniform arrival precision. This paper extends the previous concept to enhance the performance of time-based arrival management and consider mixed aircraft performance. Aircraft equipped to achieve a high degree of schedule conformance may be scheduled to the shorter path under the same conditions that a less equipped aircraft would be scheduled to the longer path, giving the equipped aircraft an advantage that can be seamlessly integrated into the scheduler. The arrival scheduler with shortcut path options for mixed aircraft performance is applied to a model of first-come first-served terminal metering at Los Angeles International Airport. Whereas clear system benefits were found for tactical shortcut routing and higher percentages of equipped aircraft, very little advantage could be seen for equipped over unequipped aircraft that could be used to incentivize early equipage.

Equipage operations↗

NASA NTRS2020

PCB Quality Metrics that Drive Reliability (PD 18)

Risk based technology infusion is a deliberate and systematic process which defines the analysis and communication methodology by which new technology is applied and integrated into existing and new designs, identifies technology development needs based on trends analysis and facilitates the identification of shortfalls against performance objectives. This presentation at IPC Works Asia Aerospace 2019 Events provides the audience a snapshot of quality variations in printed wiring board quality, as assessed, using experiences in processing and risk analysis of PWB structural integrity coupons. The presentation will focus on printed wiring board quality metrics used, the relative type and number of non-conformances observed and trend analysis using statistical methods. Trend analysis shows the top five non-conformances observed across PWB suppliers, the root cause(s) behind these non-conformance and suggestions of mitigation plans. The trends will then be matched with the current state of the PWB supplier base and its challenges and opportunities. The presentation further discusses the risk based SMA approaches and methods being applied at GSFC for evaluating candidate printed wiring board technologies which promote the adoption of higher throughput and faster processing technology for GSFC missions.

Sood, Bhanu↗

NASA NTRSDate not supplied

PCB Quality Metrics that Drive Reliability

Risk-based technology infusion is a deliberate and systematic process that defines the analysis and communication methodology by which new technology is applied and integrated into existing and new designs, identifies technology development needs based on trends analysis and facilitates the identification of shortfalls against performance objectives. This presentation at IPC Works Asia Aerospace 2021 Events provides the audience a snapshot of quality variations in printed-wiring-board quality, as assessed, using experiences in processing and risk analysis of PWB structural integrity coupons. The presentation will focus on printed-wiring-board quality metrics used, the relative type and number of non-conformances observed and trend analysis using statistical methods. Trend analysis shows the top five non-conformances observed across PWB suppliers, the root cause(s) behind these non-conformance and suggestions of mitigation plans. The trends are then matched with the current state of the PWB supplier base and its challenges and opportunities. The presentation further discusses the risk-based SMA approaches and methods being applied at GSFC for evaluating candidate printed wiring board technologies that promote the adoption of higher throughput and faster processing technology for GSFC missions.

Printed circuit boards↗

NASA NTRSDate not supplied

A Physics of Failure Approach to Evaluate Printed Circuit Board Reliability

Risk based technology infusion is a deliberate and systematic process which defines the analysis and communication methodology by which new technology is applied and integrated into existing and new designs, identifies technology development needs based on trends analysis and facilitates the identification of shortfalls against performance objectives. This presentation at IPC Works Asia Aerospace 2019 Events provides the audience a snapshot of quality variations in printed wiring board quality, as assessed, using experiences in processing and risk analysis of PWB structural integrity coupons. The presentation will focus on printed wiring board quality metrics used, the relative type and number of non-conformances observed and trend analysis using statistical methods. Trend analysis shows the top five non-conformances observed across PWB suppliers, the root cause(s) behind these non-conformance and suggestions of mitigation plans. The trends will then be matched with the current state of the PWB supplier base and its challenges and opportunities. The presentation further discusses the risk based SMA approaches and methods being applied at GSFC for evaluating candidate printed wiring board technologies which promote the adoption of higher throughput and faster processing technology for GSFC missions.

Printed Circuit Boards↗

NASA NTRSDate not supplied

SIBatt-3D: In-Space/On-Surface 3D Printing of Sodium Ion Batteries from ISRU Materials

Constructed more than 20 years ago, the International Space Station’s primary power system originally used nickel-hydrogen batteries with a lifetime of 6.5 years, until NASA began the process of replacing them in 2016 with lithium-ion batteries with a lifetime of 10 years. The demanding and costly process was accomplished after four flights of the Japanese H-II Transfer Vehicle cargo spacecraft (with a cost of about $10,000 per pound of payload), and 13 different astronauts conducting 14 spacewalks. Besides utilization in the ISS, rechargeable batteries are present in many space applications: they are installed in exploration robots, life support systems and in portable communication devices, to mention some. In this context, this project is focused on the in-space manufacturing of shape-conformable batteries using in-situ resources, and aims to address the NASA’s gaps related to the development of next generation of energy storage devices (TX03), as well as in-space manufacturing and in-situ resource utilization (TX07). The proposed work also tackles the HEOMD’s objectives targeting the in-space additive manufacturing (AM) from Lunar/Martian materials (regolith as AM feedstock) to reinvigorate America’s Human Space Exploration Program (SPD-1). This project is in direct alignment with the STMD’s objectives to demonstrate in-space autonomous manufacturing and assembly of complete systems by 2030, and to enable humans to live and explore in space and on planetary surfaces by 2040 thanks to in-space habitation, infrastructure development and in-situ resource utilization (ST1 and ST5). Manufacturing of shape conformable batteries directly in-space and using in-situ resources would also contribute to reducing the payload weight and volume (TX12) for future missions, thus reducing risk for long term Mars missions where rapid resupply is logistically infeasible. Nowadays, commercial batteries consist of stacked two-dimensional (2D) sheets, which are only manufactured in restricted geometries (cylindrical and coin cell). Evolving from conventional 2D, complex 3D battery architectures have been proven to increase the electrochemical active surface area and ion diffusion path, leading to improved areal energy density and power performance. This tendency was illustrated in our recent in-depth modeling studies by simulating a classical Ragone plot exhibiting the energy-power relationship. Our team demonstrated through modeling that a complex gyroidal 3D printed battery architecture exhibits significantly improved power performances (>150% at the current density of 6C; full discharge in 10 minutes) in comparison to a traditional 3D printed planar geometry. Motivated by these results and as the fabrication of intricate 3D battery design is only possible experimentally thanks to the geometric freedom offered by additive manufacturing (AM), our team has already initiated leveraging thermoplastic material extrusion at the laboratory scale. While 3D printing of batteries is relatively recent (2013), it has witnessed a growing interest during the last recent years, as next-generation shape-conformable 3D batteries can be co-designed with the system. Consequently, dead-volume and mass brought from Earth are minimized, in addition to improved battery performance, in alignment with the aforementioned NASA’s objectives. Further, while this project is specifically dedicated to batteries, it lends itself towards the maturation of in-space manufacturing via 3D printing using in-situ resources, stated in HEOMD and STMD goals.

In-Space Manufacturing↗

NASA NTRSDate not supplied

Simulation of Radiation-Induced DNA Damage With the Code RITRACKS

INTRODUCTION DNA damage is one of the most physiologically important effects of ionizing radiation. Clustered DNA damage events, like double-strand breaks (DSBs), have the most notable biological consequences. DNA damage types depend on both the track structure of the radiation and the spatial organization of the DNA. High linear energy transfer (LET) charged nuclei, found in galactic cosmic rays (GCR), are known to produce large numbers of complex DNA damage events. The human genome is packaged into chromatin, which can take on locus-dependent and cell type-dependent spatial conformations that correspond to epigenetic states, such as more open, extended structures in transcriptionally active chromatin. These epigenetic differences can affect DNA break patterns in response to ionizing radiation, potentially creating distinct DNA repair and signaling outcomes across the genome in different cells. MATERIAL AND METHODS The code RITRACKS (Relativistic Ion Tracks), which simulates stochastic radiation track structures and radiation chemistry, was used to model damage on isolated and histone-bound DNA by various types of ions and photons. The changes made to the code to perform radiation-induced DNA damage, and simulation results on single nucleosomes are given in our recent paper. In this work, the DNA building capabilities of RITRACKS have been extended to simulate more complex DNA structures build on the coarse-grain simulation framework meso-WLCsim. This code can sample generic chromatin fiber conformation ensembles based on the geometry of nucleosomes and mechanical properties of DNA. Using RITRACKS, we simulated the fragment length distributions (FLD) of irradiated DNA structures built using the chromatin conformations of WLCsim and obtained results representative of those obtained with Radiation-Induced Correlated Cleavage with sequencing (RICC-Seq) experiments [6]. We have also performed Fe ion and photon irradiations of K562, IMR90, BJ and RPE-1 cells at NSRL to experimentally validate results. Sample processing and data analysis are in progress and any available preliminary results will be discussed. DISCUSSION The recent updates in the code RITRACKS allow the calculation of several quantities such as the DNA damage yield and the FLD. This approach can be used to model epigenetic state-specific chromatin structure parameters to leverage the epigenetic state data available for many human cell types to infer relative DNA damage sensitivity among genomic loci.

I Plante↗

NASA NTRS1999

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Non-NASA Center↗

NASA NTRS2002

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.

Evaluation Studies↗