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BACKGROUND The availability of nitrogen (N) to plants and microbes has a major influence on the structure and function of ecosystems. Because N is an essential component of plant proteins, low N availability constrains the growth of plants and herbivores. To increase N availability, humans apply large amounts of fertilizer to agricultural systems. Losses from these systems, combined with atmospheric deposition of fossil fuel combustion products, introduce copious quantities of reactive N into ecosystems. The negative consequences of these anthropogenic N inputs—such as ecosystem eutrophication and reductions in terrestrial and aquatic biodiversity—are well documented. Yet although N availability is increasing in many locations, reactive N inputs are not evenly distributed globally. Furthermore, experiments and theory also suggest that global change factors such as elevated atmospheric CO 2 , rising temperatures, and altered precipitation and disturbance regimes can reduce the availability of N to plants and microbes in many terrestrial ecosystems. This can occur through increases in biotic demand for N or reductions in its supply to organisms. Reductions in N availability can be observed via several metrics, including lowered nitrogen concentrations ([N]) and isotope ratios (δ 15 N) in plant tissue, reduced rates of N mineralization, and reduced terrestrial N export to aquatic systems. However, a comprehensive synthesis of N availability metrics, outside of experimental settings and capable of revealing large-scale trends, has not yet been carried out. ADVANCES A growing body of observations confirms that N availability is declining in many nonagricultural ecosystems worldwide. Studies have demonstrated declining wood δ 15 N in forests across the continental US, declining foliar [N] in European forests, declining foliar [N] and δ 15 N in North American grasslands, and declining [N] in pollen from the US and southern Canada. This evidence is consistent with observed global-scale declines in foliar δ 15 N and [N] since 1980. Long-term monitoring of soil-based N availability indicators in unmanipulated systems is rare. However, forest studies in the northeast US have demonstrated decades-long decreases in soil N cycling and N exports to air and water, even in the face of elevated atmospheric N deposition. Collectively, these studies suggest a sustained decline in N availability across a range of terrestrial ecosystems, dating at least as far back as the early 20th century. Elevated atmospheric CO 2 levels are likely a main driver of declines in N availability. Terrestrial plants are now uniformly exposed to ~50% more of this essential resource than they were just 150 years ago, and experimentally exposing plants to elevated CO 2 often reduces foliar [N] as well as plant-available soil N. In addition, globally-rising temperatures may raise soil N supply in some systems but may also increase N losses and lead to lower foliar [N]. Changes in other ecosystem drivers—such as local climate patterns, N deposition rates, and disturbance regimes—individually affect smaller areas but may have important cumulative effects on global N availability. OUTLOOK Given the importance of N to ecosystem functioning, a decline in available N is likely to have far-reaching consequences. Reduced N availability likely constrains the response of plants to elevated CO 2 and the ability of ecosystems to sequester carbon. Because herbivore growth and reproduction scale with protein intake, declining foliar [N] may be contributing to widely reported declines in insect populations and may be negatively affecting the growth of grazing livestock and herbivorous wild mammals. Spatial and temporal patterns in N availability are not yet fully understood, particularly outside of Europe and North America. Developments in remote sensing, accompanied by additional historical reconstructions of N availability from tree rings, herbarium specimens, and sediments, will show how N availability trajectories vary among ecosystems. Such assessment and monitoring efforts need to be complemented by further experimental and theoretical investigations into the causes of declining N availability, its implications for global carbon sequestration, and how its effects propagate through food webs. Responses will need to involve reducing N demand via lowering atmospheric CO 2 concentrations, and/or increasing N supply. Successfully mitigating and adapting to declining N availability will require a broader understanding that this phenomenon is occurring alongside the more widely recognized issue of anthropogenic eutrophication. Intercalibration of isotopic records from leaves, tree rings, and lake sediments suggests that N availability in many terrestrial ecosystems has steadily declined since the beginning of the industrial era. Reductions in N availability may affect many aspects of ecosystem functioning, including carbon sequestration and herbivore nutrition. Shaded areas indicate 80% prediction intervals; marker size is proportional to the number of measurements in each annual mean. Isotope data: (tree ring) K. K. McLauchlan et al. , Sci. Rep. 7 , 7856 (2017); (lake sediment) G. W. Holtgrieve et al. , Science 334 , 1545–1548 (2011); (foliar) J. M. Craine et al. , Nat. Ecol. Evol. 2 , 1735–1744 (2018)more » « less
Proteins and nucleic acids participate in essentially every biochemical process in living organisms, and the elucidation of their structure and motions is essential for our understanding how these molecular machines perform their function. Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful versatile technique that provides critical information on the molecular structure and dynamics. Spin-relaxation data are used to determine the overall rotational diffusion and local motions of biological macromolecules, while residual dipolar couplings (RDCs) reveal local and long-range structural architecture of these molecules and their complexes. This information allows researchers to refine structures of proteins and nucleic acids and provides restraints for molecular docking. Several software packages have been developed by NMR researchers in order to tackle the complicated experimental data analysis and structure modeling. However, many of them are offline packages or command-line applications that require users to set up the run time environment and also to possess certain programming skills, which inevitably limits accessibility of this software to a broad scientific community. Here we present new science gateways designed for NMR/structural biology community that address these current limitations in NMR data analysis. Using the GenApp technology for scientific gateways (https://genapp.rocks), we successfully transformed ROTDIF and ALTENS, two offline packages for bio-NMR data analysis, into science gateways that provide advanced computational functionalities, cloud-based data management, and interactive 2D and 3D plotting and visualizations. Furthermore, these gateways are integrated with molecular structure visualization tools (Jmol) and with gateways/engines (SASSIE-web) capable of generating huge computer-simulated structural ensembles of proteins and nucleic acids. This enables researchers to seamlessly incorporate conformational ensembles into the analysis in order to adequately take into account structural heterogeneity and dynamic nature of biological macromolecules. ROTDIF-web offers a versatile set of integrated modules/tools for determining and predicting molecular rotational diffusion tensors and model-free characterization of bond dynamics in biomacromolecules and for docking of molecular complexes driven by the information extracted from NMR relaxation data. ALTENS allows characterization of the molecular alignment under anisotropic conditions, which enables researchers to obtain accurate local and long-range bond-vector restraints for refining 3-D structures of macromolecules and their complexes. We will describe our experience bringing our programs into GenApp and illustrate the use of these gateways for specific examples of protein systems of high biological significance. We expect these gateways to be useful to structural biologists and biophysicists as well as NMR community and to stimulate other researchers to share their scientific software in a similar way.more » « less
X-ray crystallography has been invaluable in delivering structural information about proteins. Previously, an approach has been developed that allows high-quality X-ray diffraction data to be obtained from protein crystals at and above room temperature. Here, this previous work is built on and extended by showing that high-quality anomalous signal can be obtained from single protein crystals using diffraction data collected at 220 K up to physiological temperatures. The anomalous signal can be used to directly determine the structure of a protein,more » « less
i.e.to phase the data, as is routinely performed under cryoconditions. This ability is demonstrated by obtaining diffraction data from model lysozyme, thaumatin and proteinase K crystals, the anomalous signal from which allowed their structures to be solved experimentally at 7.1 keV X-ray energy and at room temperature with relatively low data redundancy. It is also demonstrated that the anomalous signal from diffraction data obtained at 310 K (37°C) can be used to solve the structure of proteinase K and to identify ordered ions. The method provides useful anomalous signal at temperatures down to 220 K, resulting in an extended crystal lifetime and increased data redundancy. Finally, we show that useful anomalous signal can be obtained at room temperature using X-rays of 12 keV energy as typically used for routine data collection, allowing this type of experiment to be carried out at widely accessible synchrotron beamline energies and enabling the simultaneous extraction of high-resolution data and anomalous signal. With the recent emphasis on obtaining conformational ensemble information for proteins, the high resolution of the data allows such ensembles to be built, while the anomalous signal allows the structure to be experimentally solved, ions to be identified, and water molecules and ions to be differentiated. Because bound metal-, phosphorus- and sulfur-containing ions all have anomalous signal, obtaining anomalous signal across temperatures and up to physiological temperatures will provide a more complete description of protein conformational ensembles, function and energetics.
Frappier, Lori (Ed.)ABSTRACT Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo . Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8’s C-terminus and a portion of VP1’s N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae . The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.more » « less
The mimicry of protein tertiary folds by chains artificial in backbone chemical composition leads to proteomimetic analogues with potential utility as bioactive agents and as tools, to shed light on biomacromolecule behavior. Notable successes toward such molecules have been achieved; however, as protein structural diversity is vast, design principles must be continually honed as they are applied to new prototype folding patterns. One specific structure where a gap remains in understanding how to effectively generate modified backbone analogues is the metal‐binding β‐turn found in zinc finger domains. The literature precedent suggests several factors that may act in concert, including the artificial moiety used to modify the turn, the sequence in which it is applied, and modifications present elsewhere in the domain. Here, we report efforts to gain insights into these issues and leverage these insights to construct a zinc finger mimetic with backbone modifications throughout its constituent secondary structures. We first conduct a systematic comparison of four turn mimetics in a common host sequence, quantifying relative efficacy for use in a metal‐binding context. We go on to construct a proteomimetic zinc finger domain in which the helix, strands, and turn are simultaneously modified, resulting in a variant with 23% artificial residues, a tertiary fold indistinguishable from the prototype, and a folded stability comparable to the natural backbone on which the variant is based. Collectively, the results reported provide new insights into the effects of backbone modification on the structure and stability of metal‐binding domains and help inform the design of metalloprotein mimetics.