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DOE OSTI2017

Three-Dimensional Geometry of Collagenous Tissues by Second Harmonic Polarimetry

Collagen is a biological macromolecule capable of second harmonic generation, allowing label-free detection in tissues; in addition, molecular orientation can be determined from the polarization dependence of the second harmonic signal. Previously we reported that in-plane orientation of collagen fibrils could be determined by modulating the polarization angle of the laser during scanning. We have now extended this method so that out-of-plane orientation angles can be determined at the same time, allowing visualization of the 3-dimensional structure of collagenous tissues. This approach offers advantages compared with other methods for determining out-of-plane orientation. First, the orientation angles are directly calculated from the polarimetry data obtained in a single scan, while other reported methods require data from multiple scans, use of iterative optimization methods, application of fitting algorithms, or extensive post-optical processing. Second, our method does not require highly specialized instrumentation, and thus can be adapted for use in almost any nonlinear optical microscopy setup. It is suitable for both basic and clinical applications. We present three-dimensional images of structurally complex collagenous tissues that illustrate the power of such 3-dimensional analyses to reveal the architecture of biological structures.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2004

Forest Attributes from Radar Interferometric Structure and its Fusion with Optical Remote Sensing

The possibility of global, three-dimensional remote sensing of forest structure with interferometric synthetic aperture radar (InSAR) bears on important forest ecological processes, particularly the carbon cycle. InSAR supplements two-dimensional remote sensing with information in the vertical dimension. Its strengths in potential for global coverage complement those of lidar (light detecting and ranging), which has the potential for high-accuracy vertical profiles over small areas. InSAR derives its sensitivity to forest vertical structure from the differences in signals received by two, spatially separate radar receivers. Estimation of parameters describing vertical structure requires multiple-polarization, multiple-frequency, or multiple-baseline InSAR. Combining InSAR with complementary remote sensing techniques, such as hyperspectral optical imaging and lidar, can enhance vertical-structure estimates and consequent biophysical quantities of importance to ecologists, such as biomass. Future InSAR experiments will supplement recent airborne and spaceborne demonstrations, and together with inputs from ecologists regarding structure, they will suggest designs for future spaceborne strategies for measuring global vegetation structure.

InSAR↗

DOE OSTI2021

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (~68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward ( 15Z P r → 15E P g ) and reverse ( 15E P g → 15Z P r ) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15Z P r photoproduct in AnPixJg2. Altogether, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φ p within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery

Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A 2A receptor (A 2A R), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a “gatekeeper” for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1993

Wall extensibility: its nature, measurement and relationship to plant cell growth

Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.

Review, Academic↗

DOE OSTI2021

Semi-automated Design of Artificial Intelligence Earth Systems Models

Prediction and observation of water cycles at various scales involve not only patterns isolated in space and time, but also modeling of complex spatio-temporal relationships across multiple domains. For instance, evapotranspiration (ET) and leaf area indexes (LAI) are two parameters that are needed to accurately model and understand land-atmosphere processes. Accurate assessments of ET and LAI are critical for understanding hydrological processes, deforestation, crop yield, and irrigation impacts. However, ET estimates for global simulations are available at very coarse spatial resolution. They are usually derived from satellite data based on broad plant functional types (PFTs), which fail to capture fine-scale variations because of changes in vegetation type across the globe. Similarly LAI estimates have typically been derived from vegetation indices at global scales or estimated locally using physical models, both of which suffer from a range of uncertainties that impact model sensitivity. The new era of AI model development for Earth systems (ES) calls for data-driven methods that provide domain scientists with uncertainty-aware estimations of biophysical parameters such as ET and LAI in a generalizable, interpretable, and discoverable manner.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

2022 American Conference on Neutron Scattering (ACNS 2022)

The 11th American Conference on Neutron Scattering (ACNS 2022) will be held on June 5-9, 2022, in Boulder, CO. The Conference will provide essential information on the breadth and depth of current neutron-related research worldwide. Hosted by the Neutron Scattering Society of America, this year’s Conference will feature a combination of invited and contributed talks, poster sessions, and tutorials. Topics of the conference are: Advances in Neutron Facilities, Instrumentation and Software: Developments in sources, instrumentation, sample environments and control software. Hard Condensed Matter: Magnetism, correlated metals, quantum/topological materials, superconductors, ferroelectrics, multiferroics, glasses, and disorder phenomena. Submissions outlining examples of neutron scattering in industrial and engineering applications involving hard condensed matter systems are also encouraged. Soft Matter: Neutron studies of soft materials and related fields including in situ and in operando studies. Polymers, surfactants, emulsions, gels, nanoparticles, colloidal suspensions and more. Submissions of computational studies or applications of machine learning beneficial to neutron scattering experiments, as well as examples of neutron scattering in industrial and engineering applications are strongly encouraged. Biology, Biophysics and Biotechnology: Neutron studies of biological and biologically relevant systems. Proteins, bio membranes, biological assemblies, natural materials, nucleic acids, drug-delivery platforms and biomedical systems. Submissions of computational studies or applications of machine learning beneficial to biological neutron scattering experiments, as well as examples of neutron scattering in applied research involving biological systems, are strongly encouraged. Materials Chemistry and Energy: Neutron-based studies of functional materials and materials for energy applications. Examples include porous materials such as metal organic frameworks (MOFs), zeolites; phosphors; novel pigments; electrolytes; catalysts; ionic conductors/cathode materials; photovoltaic materials (hybrid perovskites); thermoelectrics; magnetocalorics/electrocalorics. Structural Materials and Engineering: Neutron scattering studies of materials and engineering processes including structural materials, concrete and metals, as well as engineering processes including combustion, corrosion, additive manufacturing, and others. Neutron Physics: Fundamental physical studies of the neutron and related areas. Emerging Applications in Neutron Scattering: Machine Learning and Data Science: Advances in computing power have contributed to rapidly evolving machine learning and data science fields that can be leveraged to the benefit of the neutron scattering community. The purpose of this session is to highlight recent advances in machine learning and data science and to serve as the foundation of a parallel data and computation track highlighting computation advances and applications in neutron scattering throughout the conference.

36 MATERIALS SCIENCE↗

DOE OSTI2016

Using landscape typologies to model socioecological systems: Application to agriculture of the United States Gulf Coast

Agricultural enterprises are vulnerable to the effects of climate variability and change. Improved understanding of the determinants of vulnerability and adaptive capacity in agricultural systems is important for projecting and managing future climate risk. At present, three analytical tools dominate methodological approaches to understanding agroecological vulnerability to climate: process-based crop models, empirical crop models, and integrated assessment models. A common weakness of these approaches is their limited treatment of socio-economic conditions and human agency in modeling agroecological processes and outcomes. This study proposes a framework that uses spatial cluster analysis to generate regional socioecological typologies that capture geographic variance in regional agricultural production and enable attribution of that variance to climatic, topographic, edaphic, and socioeconomic components. This framework was applied to historical corn production (1986-2010) in the U.S. Gulf of Mexico region as a testbed. The results demonstrate that regional socioeconomic heterogeneity is an important driving force in human dominated ecosystems, which we hypothesize, is a function of the link between socioeconomic conditions and the adaptive capacity of agricultural systems. Meaningful representation of future agricultural responses to climate variability and change is contingent upon understanding interactions among biophysical conditions, socioeconomic conditions, and human agency their incorporation in predictive models.

58 GEOSCIENCES↗

DOE OSTI2015

A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. In conclusion, MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2017

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1992

Multispectral satellite observations for arid land studies

Multispectral satellite data when properly calibrated and standardized can be used synergistically for a quantitative analysis of processes and surface characteristics, and for quantifying land surface change. Relationships among multispectral satellite data (visible reflectance, surface temperature and polarization difference of microwave emission at 37 GHz frequency) have been used to develop hypotheses concerning the relative sensitivity of these data to varied land surface characteristics, which needs to be verified by field observations. Radiative transfer models have also been developed to understand these multispectral data. Interannual variations of visible reflectance and polarization difference for the period 1982-1986 over the Sahel and the Sudan zones of Africa show a lagged response with respect to the rainfall deficit during recovery from drought, which needs to be understood in terms of biophysical parameters.

Choudhury, Bhaskar J.↗

NASA NTRS1994

Space observations for global and regional studies of the biosphere

The capability to make space-based measurements of Earth at high spatial and temporal resolutions, which would not otherwise be economically or practically feasible, became available just in time to contribute to scientific understanding of the interactive processes governing the total Earth system. Such understanding has now become essential in order to take practical steps which would counteract or mitigate the pervasive impact of the growing human population on the future habitability of the Earth. The paper reviews the rationale for using space observations for studies of climate and terrestrial ecosystems at global and regional scales, as well as the requirements for such observations for studies of climate and ecosystem dynamics. The present status of these developments is reported along with initiatives under way to advance the use of satellite observations for Earth system studies. The most important contribution of space observations is the provision of physical or biophysical parameters for models representing various components of the Earth system. Examples of such parameters are given for climatic and ecosystem studies.

Cihlar, J.↗

NASA NTRS2014

Estimating Major Ion and Nutrient Concentrations in Mangrove Estuaries in Everglades National Park Using Leaf and Satellite Reflectance

Coastal mangrove ecosystems are under duress worldwide because of urban development, sea-level rise, and climate change, processes that are capable of changing the salinity and nutrient concentration of the water utilized by the mangroves. This study correlates long-term water chemistry in mangrove environments, located in Everglades National Park,with mangrove spectral reflectance measurements made at both the leaf and canopy scales. Spectral reflectance measurements were collected using a handheld spectrometer for leaf-level measurements and Landsat 5TMdata for regional coverage. Leaf-level reflectance data were collected from three mangrove species (i.e., red, black and white mangroves) across two regions; a tall mangrove (approximately 18m) and dwarf mangrove (1- 2 m) region. The reflectance data were then used to calculate a wide variety of biophysical reflectance indices (e.g., NDVI, EVI, SAVI) to determine signs of stress. Discrete, quarterly water samples from the surface water, groundwater, and pore water (20 and 85 cm depths) and daily autonomous surface water samples were collected at each site and analyzed for major anions (Cl(−) and SO(2(-)/4)), cations (Na(+), K(+), Mg(sup 2(+)), and Ca(sup 2(+)), total nitrogen (TN) and total phosphorus (TP).Mangrove sites that exhibited the highest salinity and ionic concentrations in the surface and subsurface water also had the lowest near-infrared reflectance at both the leaf and satellite levels. Seasonal reflectance responses were measured in the near-infrared (NIR) wavelengths at both the leaf and canopy scales and were strongly correlated with nutrient and ionic concentrations in the surface and subsurface water, even though there was no significant separability between the three mangrove species. Study sites that experienced the greatest variability in surface and subsurface water ionic concentrations also exhibited the greatest fluctuations in NIR spectral reflectance. Landsat 5TM images were able to detect tall and dwarf mangroves by the differences in spectral indices (e.g., NDVI, NDWI, and EVI) because of the variability in the background conditions amongst the environments. In addition, Landsat 5TM images spanning 16 years (1993- 2009) were successfully used to estimate the seasonal variability in ionic concentrations in the surface water across the Florida Coastal mangrove ecotone. This study has shown that water chemistry can be estimated indirectly by measuring the change in spectral response at the leaf- or satellite-scale. Furthermore, the results of this research may be extrapolated to similar coastal mangrove systems throughout the Caribbean and world-wide wherever red, black, and white mangroves occur.

Mangroves↗

DOE OSTI2024

Inferring Stochastic Rates from Heterogeneous Snapshots of Particle Positions

Many imaging techniques for biological systems—like fixation of cells coupled with fluorescence microscopy—provide sharp spatial resolution in reporting locations of individuals at a single moment in time but also destroy the dynamics they intend to capture. In this study, these snapshot observations contain no information about individual trajectories, but still encode information about movement and demographic dynamics, especially when combined with a well-motivated biophysical model. The relationship between spatially evolving populations and single-moment representations of their collective locations is well-established with partial differential equations (PDEs) and their inverse problems. However, experimental data is commonly a set of locations whose number is insufficient to approximate a continuous-in-space PDE solution. Here, motivated by popular subcellular imaging data of gene expression, we embrace the stochastic nature of the data and investigate the mathematical foundations of parametrically inferring demographic rates from snapshots of particles undergoing birth, diffusion, and death in a nuclear or cellular domain. Toward inference, we rigorously derive a connection between individual particle paths and their presentation as a Poisson spatial process. Using this framework, we investigate the properties of the resulting inverse problem and study factors that affect quality of inference. One pervasive feature of this experimental regime is the presence of cell-to-cell heterogeneity. Rather than being a hindrance, we show that cell-to-cell geometric heterogeneity can increase the quality of inference on dynamics for certain parameter regimes. Altogether, the results serve as a basis for more detailed investigations of subcellular spatial patterns of RNA molecules and other stochastically evolving populations that can only be observed for single instants in their time evolution.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters

Solute transport across cell membranes is ubiquitous in biology as an essential physiological process. Secondary active transporters couple the unfavorable process of solute transport against its concentration gradient to the energetically favorable transport of one or several ions. The study of such transporters over several decades indicates that their function involves complex allosteric mechanisms that are progressively being revealed in atomistic detail. We focus on two well-characterized sodium-coupled symporters: the bacterial amino acid transporter LeuT, which is the prototype for the “gated pore” mechanism in the mammalian synaptic monoamine transporters, and the archaeal GltPh, which is the prototype for the “elevator” mechanism in the mammalian excitatory amino acid transporters. We present the evidence for the role of allostery in the context of a quantitative formalism that can reconcile biochemical and biophysical data and thereby connects directly to recent insights into the molecular structure and dynamics of these proteins. We demonstrate that, while the structures and mechanisms of these transporters are very different, the available data suggest a common role of specific models of allostery in their functions. We argue that such allosteric mechanisms appear essential not only for sodium-coupled symport in general but also for the function of other types of molecular machines in the membrane.

Chemistry↗

DOE OSTI2021

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

membrane curvature↗

DOE OSTI2024

Non-Invasive Biophysical Techniques to Monitor the Structural Plasticity of the Photosynthetic Machinery of Live Diatom Cells

The photosynthetic performance of diatoms depends largely on the organization and structural flexibility of their thylakoid membranes, the densely packed, highly organized membrane vesicles in which light reactions of photosynthesis occur. Different regulatory mechanisms that fine tune the photosynthetic functions affect the organization of the photosynthetic machinery at different levels of structural complexity, from the level of individual protein complexes to the macroarray of membrane proteins and the remodeling of the entire thylakoid membrane system. To monitor these reorganizations, non-invasive techniques are of special value. In this chapter, we focus our attention on three of these techniques, which have been demonstrated to provide unique and useful information on the structure and structural and functional plasticity of live diatom cells: (i) circular dichroism (CD) spectroscopy, which has provided unique information on the chiral (macro-)organization of protein complexes and on their rapid, reversible reorganizations, fine-tuning the light-harvesting processes, as well as on variations in the short-range excitonic interactions in the antenna complexes; (ii) small-angle neutron scattering (SANS), which has been used to determine the periodic organization of the thylakoid membranes and to monitor reversible ultrastructural changes on the time-scale of minutes, induced by variations in the environmental conditions such as changes in temperature or light intensity; and (iii) electrochromic shift absorbance transients (ΔA ECS ), a spectroscopic tool which has been shown to be capable of identifying distinct functional groups of the light-harvesting carotenoid fucoxanthin in different diatoms and in cells exposed to different light intensities. Future use of these techniques will most certainly contribute to the deeper understanding of key regulatory mechanisms of photosynthesis in diatoms.

Szabo, Milan↗

DOE OSTI2018

Biophysical characterization of actin bundles generated by the Chlamydia trachomatis Tarp effector

Highlights: • The F-actin binding domain of Tarp is sufficient to bundle filamentous actin. • Actin bundles formed by Tarp are more flexible than that of α-actinin and fascin. • Tarp mediated actin bundling is independent of actin nucleation. Chlamydia trachomatis entry into host cells is mediated by pathogen-directed remodeling of the actin cytoskeleton. The chlamydial type III secreted effector, translocated actin recruiting phosphoprotein (Tarp), has been implicated in the recruitment of actin to the site of internalization. Tarp harbors G-actin binding and proline rich domains required for Tarp-mediated actin nucleation as well as unique F-actin binding domains implicated in the formation of actin bundles. Little is known about the mechanical properties of actin bundles generated by Tarp or the mechanism by which Tarp mediates actin bundle formation. In order to characterize the actin bundles and elucidate the role of different Tarp domains in the bundling process, purified Tarp effectors and Tarp truncation mutants were analyzed using Total Internal Reflection Fluorescence (TIRF) microscopy. Our data indicate that Tarp mediated actin bundling is independent of actin nucleation and the F-actin binding domains are sufficient to bundle actin filaments. Additionally, Tarp-mediated actin bundles demonstrate distinct bending stiffness compared to those crosslinked by the well characterized actin bundling proteins fascin and alpha-actinin, suggesting Tarp may employ a novel actin bundling strategy. The capacity of the Tarp effector to generate novel actin bundles likely contributes to chlamydia's efficient mechanism of entry into human cells.

60 APPLIED LIFE SCIENCES↗