Spatially distributed cytokinins: Metabolism, signaling, and transport
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59 BASIC BIOLOGICAL SCIENCES↗
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Processes that Drove the Transition from Chemistry to Biology: Concepts and Evidence
Two properties are particularly germane to the transition from chemistry to biology. One is the emergence of complex molecules (polymers) capable of performing non-trivial functions, such as catalysis, energy transduction or transport across cell walls. The other is the ability of several functions to work in concert to provide reproductive advantage to systems hosting these functions. Biological systems exhibit these properties at remarkable levels of efficiency and accuracy in a way that appears effortless. However, dissection of these properties reveals great complexities that are involved. This opens a question: how a simple, ancestral system could have acquired the required properties? Other questions follow. What are the chances that a functional polymer emerges at random? What is the minimum structural complexity of a polymer to carry out a function at a reasonable level of efficiency? Can we identify concrete, protobiologically plausible mechanisms that yield advantageous coupling between different functions? These and similar questions are at the core of the main topic of this session: how soulless chemistry became life? Clearly, we do not have complete answers to any of these questions. However, in recent years a number of new and sometimes unexpected clues have been brought to light. Of particular interest are proteins because they are the main functional polymers in contemporary cells. The emergence of protein functions is a puzzle. It is widely accepted that a well ]defined, compact structure (fold) is a prerequisite for function. It is equally widely accepted that compact folds are rare among random amino acid polymers. Then, how did protein functionality start? According to one hypothesis well folded were preceded by their poorly folded, yet still functional ancestors. Only recently, however, experimental evidence supporting this hypothesis has been presented. In particular, a small enzyme capable of ligating two RNA fragments with the rate of 106 above background was evolved in vitro. This enzyme does not look like any contemporary protein. It is very flexible and its structure is kept together just by a single salt bridge between a charged residue and a coordinating zinc. A similar picture emerges from studies of simple transmembrane channels that mimic those in ancestral cells. Again, they are extremely flexible and do not form a conventional pore. Yet, they efficiently mediate ion transport. Studies on simple proteins that are on-going in several laboratories hold promise of revealing crucial links between chemical and biological catalysis and other ubiquitous cell functions. Interaction between composition, growth and division of protobiologically relevant vesicles and metabolic reactions that they encapsulate is an example of coupling between simple functions that promotes reproduction and evolution. Recent studies have demonstrated possible mechanisms by which vesicles might have evolved their composition from fatty acids to phospholipids, thus facilitating a number of new cellular functions. Conversely, it has been also demonstrated that an encapsulated metabolism might drive vesicle division. These are, again, examples of processes that might have driven the transition from chemistry to biology.
Pohorille, Andrew↗
Global analysis of SBP gene family in Brachypodium distachyon reveals its association with spike development
SQUAMOSA-promoter binding like proteins (SBPs/SPLs) are plant specific transcription factors targeted by miR156 and involved in various biological pathways, playing multi-faceted developmental roles. This gene family is not well characterized in Brachypodium. We identified a total of 18 SBP genes in B. distachyon genome. Phylogenetic analysis revealed that SBP gene family in Brachypodium expanded through large scale duplication. A total of 10 BdSBP genes were identified as targets of miR156. Transcript cleavage analysis of selected BdSBPs by miR156 confirmed their antagonistic connection. Alternative splicing was observed playing an important role in BdSBPs and miR156 interaction. Characterization of T-DNA Bdsbp9 mutant showed reduced plant growth and spike length, reflecting its involvement in the spike development. Expression of a majority of BdSBPs elevated during spikelet initiation. Specifically, BdSBP1 and BdSBP3 differentially expressed in response to vernalization. Differential transcript abundance of BdSBP1, BdSBP3, BdSBP8, BdSBP9, BdSBP14, BdSBP18 and BdSBP23 genes was observed during the spike development under high temperature. Co-expression network, protein–protein interaction and biological pathway analysis indicate that BdSBP genes mainly regulate transcription, hormone, RNA and transport pathways. Our work reveals the multi-layered control of SBP genes and demonstrates their association with spike development and temperature sensitivity in Brachypodium.
59 BASIC BIOLOGICAL SCIENCES↗
Strong Potential Gradients and Electron Confinement in ZnO Nanoparticle Films: Implications for Charge-Carrier Transport and Photocatalysis
We report that zinc oxide (ZnO) nanomaterials are promising components for chemical and biological sensors and photocatalytic conversion and operate as electron collectors in photovoltaic technologies. Many of these applications involve nanostructures in contact with liquids or exposed to ambient atmosphere. Under these conditions, single-crystal ZnO surfaces are known to form narrow electron accumulation layers with few nanometer spatial penetration into the bulk. A key question is to what extent such pronounced surface potential gradients can develop in the nanophases of ZnO, where they would dominate the catalytic activity by modulating charge-carrier mobility and lifetimes. Here, we follow the temperature-dependent surface electronic structure of nanoporous ZnO with photoemission spectroscopy to reveal a sizable, spatially averaged downward band bending for the hydroxylated state and a conservative upper bound of < 6 nm for the spatial extent of the associated potential gradient. This nanoscale confinement of conduction-band electrons to the nanoparticle film surface is crucial for a microscopic understanding and further optimization of charge transport and photocatalytic function in complex ZnO nanomaterials.
36 MATERIALS SCIENCE↗
A Visual Tour of Carbon Export by Sinking Particles
To better quantify the ocean's biological carbon pump, we resolved the diversity of sinking particles that transport carbon into the ocean's interior, their contribution to carbon export, and their attenuation with depth. Sinking particles collected in sediment trap gel layers from four distinct ocean ecosystems were imaged, measured, and classified. The size and identity of particles was used to model their contribution to particulate organic carbon (POC) flux. Measured POC fluxes were reasonably predicted by particle images. Nine particle types were identified, and most of the compositional variability was driven by the relative contribution of aggregates, long cylindrical fecal pellets, and salp fecal pellets. While particle composition varied across locations and seasons, the entire range of compositions was measured at a single well-observed location in the subarctic North Pacific over one month, across 500 m of depth. The magnitude of POC flux was not consistently associated with a dominant particle class, but particle classes did influence flux attenuation. Long fecal pellets attenuated most rapidly with depth whereas certain other classes attenuated little or not at all with depth. Small particles (<100 m) consistently contributed ~5% to total POC flux in samples with higher magnitude fluxes. The relative importance of these small particle classes (spherical mini pellets, short oval fecal pellets, and dense detritus) increased in low flux environments (up to 46% of total POC flux). Imaging approaches that resolve large variations in particle composition across ocean basins, depth, and time will help to better parameterize biological carbon pump models
Carbon flux↗
Substrate binding-induced conformational transitions in the omega-3 fatty acid transporter MFSD2A
Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na + -dependent uptake of ω-3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na + . Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na + -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a “trap-and-flip” mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.
59 BASIC BIOLOGICAL SCIENCES↗
Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter
Na + is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na + in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na + gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na + -dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na + , Mg 2+ , K + , and Ca 2+ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na + bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na + may be depleted to study conformational changes in the LeuT transport cycle.
59 BASIC BIOLOGICAL SCIENCES↗
Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass
Background: Switchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress. Results: In total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls. Conclusion: Our study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.
59 BASIC BIOLOGICAL SCIENCES↗
Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism
BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three “binding”-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.
59 BASIC BIOLOGICAL SCIENCES↗
Self‐assembling Peptides with Internal Ionizable Unnatural Amino Acids: A General Approach to pH‐responsive Peptide Materials
Self‐assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH‐responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH‐responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain. Through a combined experimental and computational investigation, we demonstrate that these residues can be accommodated and stabilized within the internal hydrophobic compartment of the peptide assembly. The hydrophobic microenvironment shifts their pK a significantly from a basic pH typically found for free amines to a more biologically relevant pH in the weakly acidic range. The pH‐induced ionization and ionization‐dependent self‐assembly and disassembly are thoroughly investigated and correlated with the biological activity of the assembly. This new approach has unique advantages in tuning the pH‐responsiveness of self‐assembled peptides across a large pH range in a complex biological environment. We anticipate the ionizable amino acids developed here can be widely applicable to the synthesis and self‐assembly of many amphiphilic peptides with endowed pH‐responsive properties to enhance their biological activities toward applications ranging from targeted therapeutic delivery to proton transport.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
The role of depressed metabolism in increased radio-resistance
The results of experiments on hamsters and rats to determine physiological responses to various temperature conditions are presented. The experimental methods described are considered to be applicable to future mammalian experiments in space. Renal function was examined in the golden hamster as a function of body temperature. Hamsters were also acclimated to heat and metabolic rates, body temperature, skin temperature, cardiac distribution and whole body hematocrits were measured. In addition, the effects of heat stress on the intestinal transport of sugars in the hamster and rat were studied. The biological effects of prolonged space flight and methods of simulating weightlessness are also discussed.
Musacchia, X. J.↗
An Energetic Concept of Habitability for the Deep Subsurface
Universally, life must be characterized by a characteristic level of order and complexity. In the most general sense, habitability could then be defined as the set of factors required to allow the creation and maintenance of molecular complexity. These factors are: chemical raw materials; energy with which to assemble those materials into complex molecules and sustain the resultant state of complexity; a solvent that allows the interaction of complex molecules, promotes tertiary structure, and permits compartmentalization; and environmental conditions that permit the assembly and maintenance of complex molecules. On Earth, these general requirements correspond to the major biogenic elements C, H, O, N, P, S; chemical or light energy; the solvent water; and specific ranges of temperature, pH, radiation, ionic strength, and so forth, which have thus far been determined on and exclusively empirical basis. Importantly, while the complete absence of any of these factors ensures uninhabitable conditions, the mere presence of all four does not guarantee habitability. In each case - even that of water - it is a question of degree. This question can be couched in quantitative terms by considering the impact of each of these factors on cellular energy balance. More "extreme" conditions (e.g., high temperature, high or low pH, etc.), lower water activity, and low concentrations of nutrients incur or have potential to be addressed by increased investment of energy on the part of the cell. This must be balanced by energy conservation in the cell, noting that biochemical, mass transport, and abiotic chemical limitations intervene between environmental energy availability and biological energy capture. Similarly, lower boundary conditions are emplaced on useful environmental energy yields by the "quantized" nature of biological energy conservation, and upper boundary conditions are emplaced by energy levels or fluxes that are destructive with respect to complexity. This energetic framework, with boundary conditions supplied by the specifics of the biochemistry in question, offers a generalized, yet quantitative means of assessing the habitability of any system with respect to complex life.
Hoehler, Tori M.↗
Combinatorial Multidomain Mesoporous Chips and a Method for Fractionation, Stabilization, and Storage of Biomolecules
A new fractionation device shows desirable features for exploratory screening and biomarker discovery. The constituent MSCs may be tailored for desired pore sizes and surface properties and for the sequestration and enrichment of extremely low abundant protein and peptides in desired ranges of the mass/charge spectrum. The MSCs are effective in yielding reproducible extracts from complex biological samples as small as 10 microliter in a time as short as 30 minutes. They are inexpensive to manufacture, and allow for scaled up production to attain the simultaneous processing of a large number of samples. The MSCs are multiplexed, label-free diagnostic tools with the potential of biological recognition moiety modification for enhanced specificity. The MSCs may store, protect and stabilize biological fluids, enabling the simplified and cost-effective collection and transportation of clinical samples. The MSC-based device may serve as a diagnostic tool to complement histopathology, imaging, and other conventional clinical techniques. The MSCs mediated identification of disease-specific protein signatures may help in the selection of personalized therapeutic combinations, in the real-time assessment of therapeutic efficacy and toxicity, and in the rational modulation of therapy based on the changes in the protein networks associated with the prognosis and the drug resistance of the disease.
Ferrari, Mauro↗
Bioreactor Mass Transport Studies
The objectives of the proposed research efforts were to develop both a simulation tool and a series of experiments to provide a quantitative assessment of mass transport in the NASA rotating wall perfused vessel (RWPV) bioreactor to be flown on EDU#2. This effort consisted of a literature review of bioreactor mass transport studies, the extension of an existing scalar transport computer simulation to include production and utilization of the scalar, and the evaluation of experimental techniques for determining mass transport in these vessels. Since mass transport at the cell surface is determined primarily by the relative motion of the cell assemblage and the surrounding fluid, a detailed assessment of the relative motion was conducted. Results of the simulations of the motion of spheres in the RWPV under microgravity conditions are compared with flight data from EDU#1 flown on STS-70. The mass transport across the cell membrane depends upon the environment, the cell type, and the biological state of the cell. Results from a literature review of cell requirements of several scalars are presented. As a first approximation, a model with a uniform spatial distribution of utilization or production was developed and results from these simulations are presented. There were two candidate processes considered for the experimental mass transport evaluations. The first was to measure the dissolution rate of solid or gel beads. The second was to measure the induced fluorescence of beads as a stimulant (for example hydrogen peroxide) is infused into the vessel. Either technique would use video taped images of the process for recording the quantitative results. Results of preliminary tests of these techniques are discussed.
Kleis, Stanley J.↗
Sugar transporters in grasses: Function and modulation in source and storage tissues
Carbohydrate partitioning, the process of transporting carbohydrates from photosynthetic (source) tissues, such as leaves, to non-photosynthetic (sink) tissues, such as stems, roots, and reproductive structures, is vital not only for the growth and development of plants but also for withstanding biotic and abiotic stress. In many plants, sucrose is the primary form of carbohydrate loaded into the phloem for long-distance transport and unloaded into the sink tissues for utilization or storage. Here, we highlight recent findings about 1) phloem loading in grasses, 2) the principal families of sugar transporters involved in sucrose transport, and 3) novel mechanisms by which the activities of sugar transporters are modulated. We discuss exciting discoveries from eudicot species that provide valuable insights regarding the regulation of these sugar transporters, which may be translatable to monocot species. As we better understand the intricate pathways that control the activities of various sugar transporters, we can utilize this knowledge for developing improved crop varieties.
59 BASIC BIOLOGICAL SCIENCES↗
Nanopores with dynamic pore opening diameter
Solid state nanopores have emerged as model systems for understanding transport properties on the nanoscale. They serve as templates for both preparing mimics of biological channels and designing biological sensors. Unlike their biological inspirations, the majority of nanopores prepared thus far, however, have been structurally static devices such that the pore opening diameter is fixed. If we could prepare nanopores whose opening diameter fluctuated in time with controlled amplitude at known locations in the pore, we could create ionic memristors as well as achieve new transport modes. Here we present ∼10 nm diameter single nanopores drilled through a 10 nm thick gold layer positioned on top of a 30 nm thick silicon nitride film. Two types of devices were prepared; one containing single stranded DNA and the other containing hairpin DNA attached to the discrete layer of gold using thiol chemistry. When an external electric field was applied across a nanopore with single stranded DNA, the nanoconfined DNA molecules exhibited steric and electrical constraints that led to memristor-like behavior in the current–voltage curves. The degree of hysteresis was controlled by salt concentration, magnitude of voltage and pore diameter. In contrast, nanopores containing DNA hairpins conducted similar currents in forward and reverse bias in agreement with the rigidity of the hairpin molecule. The experiments are explained by Brownian dynamics simulations that reveal voltage and salt concentration induced changes in DNA extension. The degree of DNA extension was also found to be dependent on the location of the molecules along the pore axis. The nanopores presented here provide the first steps towards preparation of non-equilibrium nanopore systems.
Vlassiouk, Ivan [ORNL] (ORCID:0000000254940386)↗
How state transitions balance photosynthetic electron transport in plants – a quantitative study
In plants, the process of state transition regulates the allocation of sunlight energy between Photosystem II (PSII) and PSI. However, the implications of state transitions for harmonizing electron transport rates between photosystems, and a full quantitative picture of this process, remain underexplored. We integrated quantitative biology (biochemical and biophysical approaches) with in vivo spectroscopy on wild-type Arabidopsis and protein phosphorylation mutants. This combination facilitated monitoring of Chl redistribution and its functional implications for light harvesting and electron transport. Our findings demonstrate the reallocation of 12% of highly phosphorylated ‘extra’ light-harvesting complex II under state 2 from stacked to unstacked thylakoids. This reduces the number of Chls per PSII from 216 to 182, while increasing the number in PSI from 187 to 223. Such Chl redistribution compensates for differences in photosystem stoichiometry and photochemical quantum efficiencies, thereby precisely synchronizing electron transport rates in both photosystems. Mutant analyses corroborate that this regulatory mechanism involves reversible phosphorylation. We inferred that state transitions optimize linear electron transport, leaving no additional capacity for cyclic electron transport. Furthermore, the results suggest that the controversies about long-range migration of LHCII from stacked to unstacked thylakoid domains arise from differences in phosphorylation levels.
59 BASIC BIOLOGICAL SCIENCES↗
Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell
Abstract Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10−9 to 10−6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.
Uhlemann, Eva-Maria E.↗