SEARCH · Engineering Papers

Results for “Biophysical Processes”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27

All sources NASA NTRS DOE OSTI

NASA NTRS1996

Strategies for "minimal growth maintenance" of cell cultures: a perspective on management for extended duration experimentation in the microgravity environment of a Space station

How cells manage without gravity and how they change in the absence of gravity are basic questions that only prolonged life on a Space station will enable us to answer. We know from investigations carried out on various kinds of Space vehicles and stations that profound physiological effects can and often to occur. We need to know more of the basic biochemistry and biophysics both of cells and of whole organisms in conditions of reduced gravity. The unique environment of Space affords plant scientists an unusual opportunity to carry out experiments in microgravity, but some major challenges must be faced before this can be done with confidence. Various laboratory activities that are routine on Earth take on special significance and offer problems that need imaginative resolution before even a relatively simple experiment can be reliably executed on a Space station. For example, scientists might wish to investigate whether adaptive or other changes that have occurred in the environment of Space are retained after return to Earth-normal conditions. Investigators seeking to carry out experiments in the low-gravity environment of Space using cultured cells will need to solve the problem of keeping cultures quiescent for protracted periods before an experiment is initiated, after periodic sampling is carried out, and after the experiment is completed. This review gives an evaluation of a range of strategies that can enable one to manipulate cell physiology and curtail growth dramatically toward this end. These strategies include cryopreservation, chilling, reduced oxygen, gel entrapment strategies, osmotic adjustment, nutrient starvation, pH manipulation, and the use of mitotic inhibitors and growth-retarding chemicals. Cells not only need to be rendered quiescent for protracted periods but they also must be recoverable and further grown if it is so desired. Elaboration of satisfactory procedures for management of cells and tissues at "near zero or minimal growth" will have great value and practical consequences for experimentation on Earth as well as in Space. All of the parameters and conditions and procedural details needed to meet all the specific objectives will be the basis of the design and fabrication of cell culture units for use in the Space environment. It is expected that this will be an evolutionary process.

STS-61A Shuttle Project↗

DOE OSTI2015

AIM for Allostery: Using the Ising Model to Understand Information Processing and Transmission in Allosteric Biomolecular Systems

In performing their biological functions, molecular machines must process and transmit information with high fidelity. Information transmission requires dynamic coupling between the conformations of discrete structural components within the protein positioned far from one another on the molecular scale. This type of biomolecular “action at a distance” is termed allostery. Although allostery is ubiquitous in biological regulation and signal transduction, its treatment in theoretical models has mostly eschewed quantitative descriptions involving the system’s underlying structural components and their interactions. Here, we show how Ising models can be used to formulate an approach to allostery in a structural context of interactions between the constitutive components by building simple allosteric constructs we termed Allosteric Ising Models (AIMs). We introduce the use of AIMs in analytical and numerical calculations that relate thermodynamic descriptions of allostery to the structural context, and then show that many fundamental properties of allostery, such as the multiplicative property of parallel allosteric channels, are revealed from the analysis of such models. The power of exploring mechanistic structural models of allosteric function in more complex systems by using AIMs is demonstrated by building a model of allosteric signaling for an experimentally well-characterized asymmetric homodimer of the dopamine D2 receptor.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

WE-AB-207B-02: A Bayesian Network Approach for Joint Prediction of Tumor Control and Radiation Pneumonitis (RP) in Non-Small-Cell Lung Cancer (NSCLC)

Purpose: NSCLC radiotherapy treatment is a trade-off between controlling the tumor while limiting radiation-induced toxicities. Here we identify hierarchical biophysical relationships that could simultaneously influence both local control (LC) and RP by using an integrated Bayesian Networks (BN) approach. Methods: We studied 79 NSCLC patients treated on prospective protocol with 56 cases of LC and 21 events of RP. Beyond dosimetric information, each patient had 193 features including 12 clinical factors, 60 circulating blood cytokines before and during radiotherapy, 62 microRNAs, and 59 single-nucleotide polymorphisms (SNPs). The most relevant biophysical predictors for both LC and RP were identified using a Markov blanket local discovery algorithm and the corresponding BN was constructed using a score-learning algorithm. The area under the free-response receiver operating characteristics (AU-FROC) was used for performance evaluation. Cross-validation was employed to guard against overfitting pitfalls. Results: A BN revealing the biophysical interrelationships jointly in terms of LC and RP was developed and evaluated. The integrated BN included two SNPs, one microRNA, one clinical factor, three pre-treatment cytokines, relative changes of two cytokines between pre and during-treatment, and gEUDs of the GTV (a=-20) and lung (a=1). On cross-validation, the AUC prediction of independent LC was 0.85 (95% CI: 0.75–0.95) and RP was 0.83 (0.73–0.92). The AU-FROC of the integrated BN to predict both LC/RP was 0.81 (0.71–0.90) based on 2000 stratified bootstrap, indicating minimal loss in joint prediction power. Conclusions: We developed a new approach for multiple outcome utility application in radiotherapy based on integrated BN techniques. The BN developed from large-scale retrospective data is able to simultaneously predict LC and RP in NSCLC treatments based on individual patient characteristics. The joint prediction is only slightly compromised compared to independent predictions. Our approach shows promise for use in clinical decision support system for personalized radiotherapy subject to multiple endpoints. These studies were supported by a grant from the NCI/NIH P01-CA59827.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Ligand placement based on prior structures: the guided ligand-replacement method

A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2025

Sustained strain applied at high rates drives dynamic tensioning in epithelial cells

Epithelial cells experience long lasting loads of different magnitudes and rates. How they adapt to these loads strongly impacts tissue health. Yet, much remains unknown about the evolution of cellular stress in response to sustained strain. Here, by subjecting cell pairs to sustained strain, we report a bimodal stress response, where in addition to the typically observed stress relaxation, a subset of cells exhibits a dynamic tensioning process with significant elevation in stress within 100 s, resembling active pulling-back in muscle fibers. Strikingly, the fraction of cells exhibiting tensioning increases with increasing strain rate. The tensioning response is accompanied by actin remodeling, and perturbation to actin abrogates it, supporting cell contractility’s role in the response. Collectively, our data show that epithelial cells adjust their tensional states over short timescales in a strain-rate dependent manner to adapt to sustained strains, demonstrating that the active pulling-back behavior could be a common protective mechanism against environmental stress.

bioinformatics↗

DOE OSTI2012

Identification and characterization of a bacterial hydrosulphide ion channel

The hydrosulphide ion (HS{sup -}) and its undissociated form, hydrogen sulphide (H{sub 2}S), which are believed to have been critical to the origin of life on Earth, remain important in physiology and cellular signalling. As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction. These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H{sub 2}S. The reduction of sulphite is a key intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella, an inducible sulphite reductase is directly linked to the regeneration of NAD{sup +}, which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite. Above a certain concentration threshold, both H{sub 2}S and HS{sup -} inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase, necessitating a release mechanism for the export of this toxic metabolite from the cell. Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS{sup -} channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate (FocA) and for nitrite (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS{sup -} ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel's crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.

08 HYDROGEN↗

DOE OSTI2022

Ligand-induced transmembrane conformational coupling in monomeric $\mathrm{EGFR}$

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

Accelerating the Whiteshell Laboratories Decommissioning Through the Implementation of a Projectized and Delivery-Focused Organization - 13074

Whiteshell Laboratories (WL) is a nuclear research site in Canada that was commissioned in 1964 by Atomic Energy of Canada Limited. It covers a total area of approximately 4,375 hectares (10,800 acres) and includes the main campus site, the Waste Management Area (WMA) and outer areas of land identified as not used for or impacted by nuclear development or operations. The WL site employed up to 1100 staff. Site activities included the successful operation of a 60 MW organic liquid-cooled research reactor from 1965 to 1985, and various research programs including reactor safety research, small reactor development, fuel development, biophysics and radiation applications, as well as work under the Canadian Nuclear Fuel Waste Management Program. In 1997, AECL made a business decision to discontinue research programs and operations at WL, and obtained government concurrence in 1998. The Nuclear Legacy Liabilities Program (NLLP) was established in 2006 by the Canadian Government to remediate nuclear legacy liabilities in a safe and cost effective manner, including the WL site. The NLLP is being implemented by AECL under the governance of a Natural Resources Canada (NRCan)/AECL Joint Oversight Committee (JOC). Significant progress has since been made, and the WL site currently holds the only Canadian Nuclear Safety Commission (CNSC) nuclear research site decommissioning license in Canada. The current decommissioning license is in place until the end of 2018. The present schedule planned for main campus decommissioning is 30 years (to 2037), followed by institutional control of the WMA until a National plan is implemented for the long-term management of nuclear waste. There is an impetus to advance work and complete decommissioning sooner. To accomplish this, AECL has added significant resources, reorganized and moved to a projectized environment. This presentation outlines changes made to the organization, the tools implemented to foster projectization, and the benefits and positive impacts on schedule and delivery. A revised organizational structure was implemented in two phases, starting 2011 April 1, to align WL staff with the common goal of decommissioning the site through the direction of the WL Decommissioning Project General Manager. On 2011 September 1, the second phase of the reorganization was implemented and WL Decommissioning staff was organized under five Divisions: Programs and Regulatory Compliance, General Site Services, Decommissioning Strategic Planning, Nuclear Facilities and Project Delivery. A new Mission, Vision and Objectives were developed for the project, and several productivity enhancements are being implemented. These include the use of an integrated and fully re-sourced Site Wide Schedule that is updated and reviewed at Plan-of-the-Week meetings, improved work distribution throughout the year, eliminating scheduling 'push' mentality, project scoreboards, work planning implementation, lean practices and various process improvement initiatives. A revised Strategic Plan is under development that reflects the improved project delivery capabilities. As a result of these initiatives, and a culture change towards a projectized approach, the decommissioning schedule will be advanced by approximately 10 years. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2017

Skeletal Dysplasia Mutations Effect on Human Filamins’ Structure and Mechanosensing

Cells’ ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered molecular dynamics simulations. The effects of skeletal dysplasia associated mutations of the structure and mechanosensing properties of Filamin were studied by combining various experimental and theoretical techniques. The results showed that Larsen syndrome associated mutations destabilize or even unfold domain 17. Interestingly, those Filamin functions that are mediated via domain 17 interactions with other proteins are not necessarily affected as strongly interacting peptide binding to mutated domain 17 induces at least partial domain folding. Mutation associated to Frontometaphyseal dysplasia, in turn, transforms 16–17 fragment from compact to an elongated form destroying the force-regulated domain pair.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2024

Applications of ArcticDEM for measuring volcanic dynamics, landslides, retrogressive thaw slumps, snowdrifts, and vegetation heights

Topographical changes are of fundamental interest to a wide range of Arctic science disciplines faced with the need to anticipate, monitor, and respond to the effects of climate change, including geohazard management, glaciology, hydrology, permafrost, and ecology. This study demonstrates several geomorphological, cryospheric, and biophysical applications of ArcticDEM – a large collection of publicly available, time-dependent digital elevation models (DEMs) of the Arctic. Our study illustrates ArcticDEM's applicability across different disciplines and five orders of magnitude of elevation derivatives, including measuring volcanic lava flows, ice cauldrons, post-failure landslides, retrogressive thaw slumps, snowdrifts, and tundra vegetation heights. We quantified surface elevation changes in different geological settings and conditions using the time series of ArcticDEM. Following the 2014–2015 Bárðarbunga eruption in Iceland, ArcticDEM analysis mapped the lava flow field, and revealed the post-eruptive ice flows and ice cauldron dynamics. The total dense-rock equivalent (DRE) volume of lava flows is estimated to be (1431 ± 2) million m 3 . Then, we present the aftermath of a landslide in Kinnikinnick, Alaska, yielding a total landslide volume of (400 ± 8) × 103 m 3 and a total area of 0.025 km 2 . ArcticDEM is further proven useful for studying retrogressive thaw slumps (RTS). The ArcticDEM-mapped RTS profile is validated by ICESat-2 and drone photogrammetry resulting in a standard deviation of 0.5 m. Volume estimates for lake-side and hillslope RTSs range between 40,000 ± 9000 m 3 and 1,160,000 ± 85,000 m 3 , highlighting applicability across a range of RTS magnitudes. A case study for mapping tundra snow demonstrates ArcticDEM's potential for identifying high-accumulation, late-lying snow areas. The approach proves effective in quantifying relative snow accumulation rather than absolute values (standard deviation of 0.25 m, bias of −0.41 m, and a correlation coefficient of 0.69 with snow depth estimated by unmanned aerial systems photogrammetry). Furthermore, ArcticDEM data show its feasibility for estimating tundra vegetation heights with a standard deviation of 0.3 m (no bias) and a correlation up to 0.8 compared to the light detection and ranging (LiDAR). The demonstrated capabilities of ArcticDEM will pave the way for the broad and pan-Arctic use of this new data source for many disciplines, especially when combined with other imagery products. The wide range of signals embedded in ArcticDEM underscores the potential challenges in deciphering signals in regions affected by various geological processes and environmental influences.

Chunli Dai↗

DOE OSTI2019

Time-resolved serial femtosecond crystallography at the European XFEL

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. In this paper, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Molecular simulations and NMR reveal how lipid fluctuations affect membrane mechanics

Lipid bilayers form the main matrix of functional cell membranes, and their dynamics underlie a host of physical and biological processes. Here we show that elastic membrane properties and collective molecular dynamics (MD) are related by the mean-square amplitudes (order parameters) and relaxation rates (correlation times) of lipid acyl chain motions. We performed all-atom MD simulations of liquid-crystalline bilayers that allow direct comparison with carbon-hydrogen (CH) bond relaxations measured with NMR spectroscopy. Previous computational and theoretical approaches have assumed isotropic relaxation, which yields inaccurate description of lipid chain dynamics and incorrect data interpretation. Instead, the new framework includes a fixed bilayer normal (director axis) and restricted anisotropic motion of the CH bonds in accord with their segmental order parameters, enabling robust validation of lipid force fields. Simulated spectral densities of thermally excited CH bond fluctuations exhibited well-defined spin-lattice (Zeeman) relaxations analogous to those in NMR measurements. Their frequency signature could be fit to a simple power-law function, indicative of nematic-like collective dynamics. Moreover, calculated relaxation rates scaled as the squared order parameters yielding an apparent K C modulus for bilayer bending. Our results show a strong correlation with K C values obtained from solid-state NMR studies of bilayers without and with cholesterol as validated by neutron spin-echo measurements of membrane elasticity. The simulations uncover a critical role of interleaflet coupling in membrane mechanics and thus provide important insights into molecular sites of emerging elastic properties within lipid bilayers.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Biomembrane Structure and Material Properties Studied With Neutron Scattering

Cell membranes and their associated structures are dynamical supramolecular structures where different physiological processes take place. Detailed knowledge of their static and dynamic structures is therefore needed, to better understand membrane biology. The structure–function relationship is a basic tenet in biology and has been pursued using a range of different experimental approaches. In this review, we will discuss one approach, namely the use of neutron scattering techniques as applied, primarily, to model membrane systems composed of lipid bilayers. An advantage of neutron scattering, compared to other scattering techniques, is the differential sensitivity of neutrons to isotopes of hydrogen and, as a result, the relative ease of altering sample contrast by substituting protium for deuterium. This property makes neutrons an ideal probe for the study of hydrogen-rich materials, such as biomembranes. In this review article, we describe isotopic labeling studies of model and viable membranes, and discuss novel applications of neutron contrast variation in order to gain unique insights into the structure, dynamics, and molecular interactions of biological membranes. We specifically focus on how small-angle neutron scattering data is modeled using different contrast data and molecular dynamics simulations. We also briefly discuss neutron reflectometry and present a few recent advances that have taken place in neutron spin echo spectroscopy studies and the unique membrane mechanical data that can be derived from them, primarily due to new models used to fit the data.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Proton transfer reactions in donor site mutants of ESR, a retinal protein from Exiguobacterium sibiricum

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. Here this two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2012

Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4;#8201;Å

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 {angstrom}. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Structures of N-terminally processed KRAS provide insight into the role of N-acetylation

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg 2+ -free structures of KRAS with flexible N-termini. In the Mg 2+ -free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg 2+ -free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg 2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg 2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg 2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1989

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such "creep" is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q10 (3.8) between 20 degrees and 30 degrees C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q10 (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu2+, Hg2+ and Al3+ were strongly inhibitory whereas most other cations, including Ca2+, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu2+, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.

NASA Program Space Biology↗