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At least 1,531 records · Page 85
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Development of Technology for Detachment of Calandria Tube Rolled Joint of Pressurised Heavy Water Reactors

Coolant channel assembly of PHWRs consisting mainly of pressure tube and calandria tube undergoes sag due to irradiation enhanced creep during its life time. Typical service life of the pressure tube is limited to about 15 full power years due to such ageing related degradation. On account of the degradation the pressure tube needs replacement more than twice during the licensed period of reactor operation. As the sag in the calandria tube is also excessive, it has to be replaced to facilitate installation of new and straight pressure tube during retubing and to avoid interference of sagged calandria tube with horizontal reactivity devices. The calandria tube and the tube sheet are joined by sandwich type rolled joints. For detaching the joint an improved technique has been developed that helps to detach the joint without affecting the groove geometry and integrity of the tube sheet. The system works on the principle of fast thermal cycle of heating and cooling of the insert and calandria tube in the rolled joint region while applying axial load to the calandria tube. Though rolled joint detachment by induction heating technique has been in use in CANDU reactors for removal of pressure tubes and calandria tubes, the present design incorporates a liquid nitrogen for faster cooling and simultaneous application of load with a gripping module for detachment and removal of calandria tube and the insert from the tube sheet of end shield in shorter time. This paper describes the methodology of rolled joint detachment, challenges involved in the design, features of the detachment system, findings of experimental trials and observations.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗
DOE OSTI2011

Recovery of phosphonate surface contaminants from glass using a simple vacuum extractor with a solid-phase microextraction fiber

Recovery of chemical contaminants from fixed surfaces for analysis can be challenging particularly if it is not possible to acquire a solid sample. A simple device is described that collects semivolatile organic compounds from fixed surfaces by creating an enclosed volume over the surface, then generating a modest vacuum. A solid-phase microextraction fiber is then inserted into the evacuated volume where it functions to sorb volatilized organic contaminants. The device is based on a syringe modified with a seal that is used to create the vacuum, with a perforable plunger through which the SPME fiber is inserted. The vacuum speeds partitioning of the semivolatile compounds into the gas phase, and reduces the boundary layer around the SPME fiber, which enables a fraction of the volatilized organics to partition into the SPME fiber. After sample collection the SPME fiber is analyzed using conventional gas chromatography/mass spectrometry. The methodology has been used to collect organophosphorus compounds from glass surfaces, to provide a simple test for the functionality of the devices. Thirty minute sampling times (deltaTvac) resulted in fractional recovery efficiencies ranged from 10(-3) to > 10(-1), and in absolute terms collection of low nanograms was demonstrated. Fractional recovery values were correlated to the vapor pressure of the compounds being sampled. Fractional recovery increased with increasing deltaTvac, and displayed a roughly logarithmic profile indicating that an operational equilibrium is being approached. Fractional recovery decreased with increasing time between exposure and sampling, however recordable quantities of the phosphonates could be collected three weeks after exposure.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Sensitivity/uncertainty analyses comparing LR-0 reactor experiments containing FLiBe salt with models for molten-salt-cooled and molten-salt-fueled reactors

Here, critical experiments using an insertion zone with FLiBe salt performed at the LR-0 reactor at Research Centre Rež (RC Rež) have been compared to application models for solid-fueled, fluoride-salt-cooled high-temperature reactor (FHR) and liquid-fueled molten salt reactor (MSR) concepts using sensitivity and uncertainty (S/U) analysis techniques. These experiments support FHR and MSR advanced reactor concepts by informing on neutron spectral effects and nuclear data uncertainties related to fluoride salts. The FLiBe salt in the LR-0 experiments is from the Oak Ridge National Laboratory Molten Salt Reactor Experiment and is enriched to greater than 99.99% 7 Li. This work is part of a broader collaboration between the United States and the Czech Republic on MSR and FHR technology. Results from the S/U analyses for FHR and MSR models indicated the most significant potential source of eigenvalue bias due to nuclear data within the FLiBe salt is radiative capture in 7 Li. Other smaller but potentially significant contributions come from 19 F, 6 Li, and other 7 Li reactions. Similarity comparisons of the RC Rež LR-0 experiments and FHR and MSR application models indicate that the LR-0 FLiBe experiments could be useful as candidate benchmarks for low-enriched uranium–fueled application models. However, sensitivity differences observed included both spectral effects, due to the LR-0 reactor being moderated by water instead of graphite and fluoride salt, and sensitivity magnitude effects driven by the amount of FLiBe inserted and its location. New experiments with refined designs to increase the volume and importance of salt in the system could provide improved data. Results also demonstrate that salt systems using thorium and/or 233 U fuel will require additional experiments with relevant driver fuels. Increased contributions from the graphite moderator and 19 F and covariance between reactions such as 233 U fission and 233 U radiative capture indicate higher uncertainty contributions from the salt and significant differences in contributions from the fuel species due to underlying uncertainties in their nuclear data.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
DOE OSTI2018

Verification of iterative matrix solutions for multipoint kinetics equations

Multipoint kinetic equation systems have been solved numerically using matrix algebra software and 1st to 4th order implicit schemes based on single-step matrix propagators. These matrix schemes have been validated successfully on demanding point kinetics benchmarks with various prescribed reactivity insertions. Verification tests have also been performed on a simple 3-region fast reactor core model with asymmetric step or ramp reactivity insertions and cross-checked with the multipoint kinetics SACRE code developed at INL and based on existing and validated stiff ODE solver packages. Accurate results can be obtained at a limited time expense. The reason for intriguing results obtained in some transients has been elucidated and linked to the multipoint matrix coefficient interpolation method during the transient.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
DOE OSTI2015

Design guidelines for avoiding thermo-acoustic oscillations in helium piping systems

Thermo-acoustic oscillations are a commonly observed phenomenon in helium cryogenic systems, especially in tubes connecting hot and cold areas. The open ends of these tubes are connected to the lower temperature (typically at 4.2 K), and the closed ends of these tubes are connected to the high temperature (300K). Cryogenic instrumentation installations provide ideal conditions for these oscillations to occur due to the steep temperature gradient along the tubing. These oscillations create errors in measurements as well as an undesirable heat load to the system. The work presented here develops engineering guidelines to design oscillation-free helium piping. This work also studies the effect of different piping inserts and shows how the proper geometrical combinations have to be chosen to avoid thermo-oscillations. The effect of an 80 K intercept is also studied and shows that thermo-oscillations can be dampened by placing the intercept at an appropriate location.

43 PARTICLE ACCELERATORS↗
DOE OSTI2012

Interaction of the Full-length Bax Protein with Biomimetic Mitochondrial Liposomes: A Small-Angle Neutron Scattering and Fluorescence Study

In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50 nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-toliposome ratio the binding seemed to saturate at about 15 Bax proteins per 50 nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop

The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18 has indicated by a stable |137 mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5 h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Furthermore, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Construction and immunological evaluation of truncated hepatitis B core particles carrying HBsAg amino acids 119–152 in the major immunodominant region (MIR)

Highlights: •The conformational HBV neutralization antigen domain was successfully displayed on the surface of truncated HBc particles. •Appropriate dialysis procedures to support the renaturing environment for the protein refolding. •Efficient purification procedures to obtain high purity and icosahedral particles of mosaic HBV antigen. •Strong immune responses not only including neutralization antibody response but also Th1 cell response were induced in mice. -- Abstract: Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenic determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp{sup 78} and Pro{sup 79}. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH{sub 4}){sub 2}SO{sub 4} precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Live-cell topology assessment of URG7, MRP6{sub 102} and SP-C using glycosylatable green fluorescent protein in mammalian cells

Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6{sub 102}, and SP-C was determined by gGFP tagging in vivo. - Abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6{sub 102}, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C{sub in} form. MRP6{sub 102} and SP-C(Leu/Val) are inserted into the membrane in C{sub out} form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. {sup 75}Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher K{sub m} than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. - Highlights: • The first characterization of a selenoprotein Trx is presented. • The selenoenzyme Trx exhibits 10-fold higher catalytic activity than Cys homologues. • Se utilization in Trx is primarily due to the catalytic advantage provided by Sec residue.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The role of receptor topology in the vitamin D3 uptake and Ca{sup 2+} response systems

The steroid hormone, vitamin D{sub 3}, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D{sub 3} and a second receptor, importin-4, imports the VDR-vitamin D{sub 3} complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ({sup 327}E – D{sup 342}), two TM “half-helix” ({sup 264}K − N{sup 276}), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ({sup 226}E – L{sup 251}; {sup 768}V – G{sup 783}; {sup 876}S – A{sup 891}) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca{sup 2+} channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D{sub 3} binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca{sup 2+} response systems and vitamin D{sub 3} uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D{sub 3}-VDR-importin-4 complex into the nucleus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Novel calcium recognition constructions in proteins: Calcium blade and EF-hand zone

Metal ions can regulate various cell processes being first, second or third messengers, and some of them, especially transition metal ions, take part in catalysis in many enzymes. As an intracellular ion, Ca{sup 2+} is involved in many cellular functions from fertilization and contraction, cell differentiation and proliferation, to apoptosis and cancer. Here, we have identified and described two novel calcium recognition environments in proteins: the calcium blade zone and the EF-hand zone, common to 12 and 8 different protein families, respectively. Each of the two environments contains three distinct structural elements: (a) the well-known characteristic Dx[DN]xDG motif; (b) an adjacent structurally identical segment, which binds metal ion in the same way between the calcium blade zone and the EF-hand zone; and (c) the following structurally variable segment, which distinguishes the calcium blade zone from the EF-hand zone. Both zones have sequence insertions between the last residue of the zone and calcium-binding residues in positions V or VI. The long insertion often connects the active and the calcium-binding sites in proteins. Using the structurally identical segments as an anchor, we were able to construct the classical calmodulin type EF-hand calcium-binding site out of two different calcium-binding motifs from two unrelated proteins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Induction of α6 and β1 integrins by acupuncture stimulation in rats

Acupuncture therapy is performed by applying the needle insertion at discrete cutaneous locations and used for the treatments of diverse symptoms and disorders. In order to elucidate mechanistic basis on how acupuncture stimulation (AS) produces therapeutic effects, it is primarily important to understand tissue responses locally at the acupuncture site (acupoint). Here, we investigated integrin protein as molecular target responding to and integrating AS. Signals of α6 and β1 integrins were clearly induced at zusanli acupoint 24 h after AS in areas of nuclear clusters around the needle track. Induction levels of integrin were largely reduced by needle insertion at non-acupuncture point or without needle rotation. Phospho-Erk1/2 was initially decreased below the basal level after AS but increased 24 h later. Induction pattern of phospho-Erk1/2 was as similar as that of α6 integrin in its selectivity to needling procedure and tissue distribution. We further found that mRNA expression of P2X3 purinergic receptor was upregulated in the dorsal root ganglion (DRG) after AS, but decreased by the inhibition of Erk1/2 activity at the acupuncture area. Moreover, AS-mediated integrin activation was required for Erk1/2 activation at the acupuncture site and regulation of pain sensitivity in the hind paw. The present results provide a new evidence on acupuncture-specific tissue response in terms of integrin induction, and further suggest that integrin activation may be involved in transmitting mechanosensory signals from the acupoint to afferent nerve fiber. - Highlights: • We investigated local tissue responses after acupuncture stimulation (AS) in rats. • AS strongly induced α6 and β1 integrins and activated Erk1/2. • Integrin Induction was primarily specific to acupuncture site and needle rotation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome

Highlights: • Expressing pglB from the genome creates a “plug and play” E. coli strain for homogeneous recombinant glycoprotein production. • This engineered strain can increase glycosylation efficiency and overall glycoprotein titres. • Parallel Reaction Monitoring can simultaneously confirm glycan addition and quantify absolute glycoprotein titres. Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Generation of splice switching oligonucleotides targeting the Cockayne syndrome group B gene product in order to change the diseased cell state

Highlights: • We designed and synthesized 24 SSO sequences to skip exon 3 in CSB mRNA. • SSO generated an additional nonsense mutation at the upstream region of PGBD3. • Such SSO up-regulated several gene expression levels in CS cells. • SSO treatment might allow CS cells to mimic UV{sup S}S cell. Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UV{sup S}S), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UV{sup S}S patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H{sub 2}O{sub 2} treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structural motifs in the RGS RZ subfamily combine to attenuate interactions with Gα subunits

Highlights: • RGS RZ subfamily specificity is set by four family-specific structural motifs. • These motifs are predicted to attenuate interactions with Gα subunits. • Insertion of these motifs into a high-activity RGS domain impaired its activity. • The motifs combine to affect activity in a non-additive manner. Regulators of G-protein Signaling (RGS) proteins inactivate heterotrimeric G proteins, thereby setting the duration of active signaling. In particular, the RGS RZ subfamily, which consists of RGS17, RGS19, and RGS20, mediates numerous physiological functions and human pathologies – mostly by functioning as GTPase Activating Proteins (GAPs) towards the Gα{sub i} subfamily. Yet, which RZ subfamily members mediate particular functions and how their GAP activity and specificity are governed at the amino acid level is not well understood. Here, we show that all RZ subfamily members have similar and relatively low GAP activity towards Gα{sub o}. We characterized four RZ-specific structural motifs that mediate this low activity, and suggest they perturb optimal interactions with the Gα subunit. Indeed, inserting these RZ-specific motifs into the representative high-activity RGS16 impaired GAP activity in a non-additive manner. Our results provide residue-level insights into the specificity determinants of the RZ subfamily, and enable to study their interactions in signaling cascades by using redesigned mutants such as those presented in this work.

60 APPLIED LIFE SCIENCES↗