53 research outputs found
Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution
Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, âfootprintingâ studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver
The XFP (17-BM) Beamline for X-ray Footprinting at NSLS-II
Hydroxyl-radical mediated synchrotron X-ray footprinting (XF) is a powerful solution-state technique in structural biology for the study of macromolecular structure and dynamics of proteins and nucleic acids, with several synchrotron resources available to serve the XF community worldwide. The XFP (Biological X-ray Footprinting) beamline at the NSLS-II was constructed on a three-pole wiggler source at 17-BM to serve as the premier beamline for performing this technique, providing an unparalleled combination of high flux density broadband beam, flexibility in beam morphology, and sample handling capabilities specifically designed for XF experiments. The details of beamline design, beam measurements, and science commissioning results for a standard protein using the two distinct XFP endstations are presented here. XFP took first light in 2016 and is now available for general user operations through peer-reviewed proposals. Currently, beam sizes from 450 Îźm Ă 120 Îźm to 2.7 mm Ă 2.7 mm (FWHM) are available, with a flux of 1.6 Ă 1016 photons s-1 (measured at 325 mA ring current) in a broadband (5-16 keV) beam. This flux is expected to rise to 2.5 Ă 1016 photons s-1 at the full NSLS-II design current of 500 mA, providing an incident power density of \u3e500 W mm-2 at full focus
A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments
Abstract Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac) and electron transport-limited (Aj) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research
The METEX study: Methotrexate versus expectant management in women with ectopic pregnancy: A randomised controlled trial
Background: Patients with ectopic pregnancy (EP) and low serum hCG concentrations and women with a pregnancy of unknown location (PUL) and plateauing serum hCG levels are commonly treated with systemic methotrexate (MTX). However, there is no evidence that treatment in these particular subgroups of women is necessary as many of these early EPs may resolve spontaneously. The aim of this study is whether expectant management in women with EP or PUL and with low but plateauing serum hCG concentrations is an alternative to MTX treatment in terms of treatment success, future pregnancy, health related quality of life and costs. Methods/Design: A multicentre randomised controlled trial in TheNetherlands. Hemodynamically stable patients with an EP visible on transvaginal ultrasound and a plateauing serum hCG concentration < 1,500 IU/L or with a persisting PUL with plateauing serum hCG concentrations < 2,000 IU/L are eligible for the trial. Patients with a viable EP, signs of tubal rupture/abdominal bleeding, or a contra-indication for MTX will not be included. Expectant management is compared with systemic MTX in a single dose intramuscular regimen (1 mg/ kg) in an outpatient setting. Serum hCG levels are monitored weekly; in case of inadequately declining, systemic MTX is installed or continued. In case of hemodynamic instability and/or signs of tubal rupture, surgery is performed. The primary outcome measure is an uneventful decline of serum hCG to an undetectable level by the initial intervention. Secondary outcomes are (re)interventions (additional systemic MTX injections and/or surgery), treatment complications, health related quality of life, financial costs, and future fertility. Analysis is performed according to the intention to treat principle. Quality of life is assessed by questionnaires before and at three time points after randomisation. Costs are expressed as direct costs with data on costs and used resources in the participating centres. Fertility is assessed by questionnaires after 6, 12, 18 and 24 months. Patients' preferences will be assessed using a discrete choice experiment. Discussion: This trial will provide guidance on the present management dilemmas in women with EPs and PULs with low and plateauing serum hCG concentrations
Global variability in leaf respiration in relation to climate, plant functional types and leaf traits
⢠Leaf dark respiration (Rdark) is an important yet poorly quantified component of the global carbon cycle. Given this, we analyzed a new global database of Rdark and associated leaf traits.
⢠Data for 899 species were compiled from 100 sites (from the Arctic to the tropics). Several woody and nonwoody plant functional types (PFTs) were represented. Mixed-effects models were used to disentangle sources of variation in Rdark.
⢠Area-based Rdark at the prevailing average daily growth temperature (T) of each site increased only twofold from the Arctic to the tropics, despite a 20°C increase in growing T (8â28°C). By contrast, Rdark at a standard T (25°C, Rdark25) was threefold higher in the Arctic than in the tropics, and twofold higher at arid than at mesic sites. Species and PFTs at cold sites exhibited higher Rdark25 at a given photosynthetic capacity (Vcmax25) or leaf nitrogen concentration ([N]) than species at warmer sites. Rdark25 values at any given Vcmax25 or [N] were higher in herbs than in woody plants.
⢠The results highlight variation in Rdark among species and across global gradients in T and aridity. In addition to their ecological significance, the results provide a framework for improving representation of Rdark in terrestrial biosphere models (TBMs) and associated land-surface components of Earth system models (ESMs)
Formation of cobaltâoxygen intermediates by dioxygen activation at a mononuclear nonheme cobalt(II) center
A mononuclear nonheme cobalt(II) complex, [(TMG3tren)CoII(OTf)](OTf) (1), activates dioxygen in the presence of hydrogen atom donor substrates, such as tetrahydrofuran and cyclohexene, resulting in the generation of a cobalt(II)-alkylperoxide intermediate (2), which then converts to the previously reported cobalt(IV)-oxo complex, [(TMG3tren)CoIV(O)]2+-(Sc(OTf)3)n (3), in >90% yield upon addition of a redox-inactive metal ion, Sc(OTf)3. Intermediates 2 and 3 represent the cobalt analogues of the proposed iron(II)-alkylperoxide precursor that converts to an iron(IV)-oxo intermediate via OâO bond heterolysis in pterin-dependent nonheme iron oxygenases. In reactivity studies, 2 shows an amphoteric reactivity in electrophilic and nucleophilic reactions, whereas 3 is an electrophilic oxidant. To the best of our knowledge, the present study reports the first example showing the generation of cobaltâoxygen intermediates by activating dioxygen at a cobalt(II) center and the reactivities of the cobaltâoxygen intermediates in oxidation reaction
Optimized Trifluoromethylation and OH Radical Labeling with Radiolysis Provide Two Fold Enhancement in the Resolution of Hydroxyl Radical Protein Footprinting
Hydroxyl radical based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in a wide range of solution conditions. In hydroxyl radical protein footprinting with X-rays (XFP), hydroxyl radicals (â˘OH) generated by water radiolysis covalently label multiple amino acid (AA) side chains simultaneously. However, HRPF technologies faces challenges in achieving their full potential due to the broad (>103) dynamic range of AAâs reactivity to â˘OH and the difficulty to detect slightly modified residues, particularly in peptides with highly reactive residues like methionine-containing peptides and in peptides containing all low reactive residues. To overcome this limitation, we developed a synchrotron-based multiplex labeling chemistry that utilizes CF3 radicals (â˘CF3) produced from a trifluoromethylation (TFM) reagent under controlled and optimized â˘OH doses generated by X-rays. We optimized the dual â˘CF3/â˘OH chemistry in this TFM labeling approach using six model peptides and lysozyme, there-by extending the existing â˘OH labeling platform with simultaneous â˘CF3 labeling. This optimization led to a two-fold increase in labeled AAs in multiplex TFM labeling, primarily by labeling to a greater degree AAs with low â˘OH reactivity via the â˘CF3 channel, while moderate and highly â˘OH reactive AAs were labeled in both â˘CF3 and â˘OH channels. Importantly, the low reactivity of methionine to â˘CF3 enabled the detection and quantification of additional AAs labeled by â˘CF3 across methionine-containing pep-tides. Consistent with observations in model peptides and protein, we observed a balanced dual â˘CF3/â˘OH chemistry and more uniform labeling of residues in both â˘CF3/â˘OH channels optimizing protein footprinting. Furthermore, the solvent accessibility of lysozyme residues directly correlated with â˘CF3 labeling demonstrating that multiplex labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF
Tuning the Reactivity of Terminal Nickel(III)âOxygen Adducts for CâH Bond Activation
Two
metastable Ni<sup>III</sup> complexes, [Ni<sup>III</sup>(OAc)Â(L)]
and [Ni<sup>III</sup>(ONO<sub>2</sub>)Â(L)] (L = <i>N</i>,<i>N</i>â˛-(2,6-dimethylphenyl)-2,6-pyridinedicarboxamidate,
OAc = acetate), were prepared, adding to the previously prepared [Ni<sup>III</sup>(OCO<sub>2</sub>H)Â(L)], with the purpose of probing the
properties of terminal late-transition metal oxidants. These high-valent
oxidants were prepared by the one-electron oxidation of their Ni<sup>II</sup> precursors ([Ni<sup>II</sup>(OAc)Â(L)]<sup>â</sup> and [Ni<sup>II</sup>(ONO<sub>2</sub>)Â(L)]<sup>â</sup>) with
trisÂ(4-bromophenyl)Âammoniumyl hexachloroantimonate. Fascinatingly,
the reaction between any [Ni<sup>II</sup>(X)Â(L)]<sup>â</sup> and NaOCl/acetic acid (AcOH) <i>or</i> cerium ammonium
nitrate ((NH<sub>4</sub>)<sub>2</sub>[Ce<sup>IV</sup>(NO<sub>3</sub>)<sub>6</sub>], CAN), yielded [Ni<sup>III</sup>(OAc)Â(L)] and [Ni<sup>III</sup>(ONO<sub>2</sub>)Â(L)], respectively. An array of spectroscopic
characterizations (electronic absorption, electron paramagnetic resonance,
X-ray absorption spectroscopies), electrochemical methods, and computational
predictions (density functional theory) have been used to determine
the structural, electronic, and magnetic properties of these highly
reactive metastable oxidants. The Ni<sup>III</sup>-oxidants proved
competent in the oxidation of phenols (weak OâH bonds) and
a series of hydrocarbon substrates (some with strong CâH bonds).
Kinetic investigation of the reactions with di-<i>tert</i>-butylphenols showed a 15-fold enhanced reaction rate for [Ni<sup>III</sup>(ONO<sub>2</sub>)Â(L)] compared to [Ni<sup>III</sup>(OCO<sub>2</sub>H)Â(L)] and [Ni<sup>III</sup>(OAc)Â(L)], demonstrating the effect
of electron-deficiency of the O-ligand on oxidizing power. The oxidation
of a series of hydrocarbons by [Ni<sup>III</sup>(OAc)Â(L)] was further
examined. A linear correlation between the rate constant and the bond
dissociation energy of the CâH bonds in the substrates was
indicative of a hydrogen atom transfer mechanism. The reaction rate
with dihydroanthracene (<i>k</i><sub>2</sub> = 8.1 M<sup>â1</sup> s<sup>â1</sup>) compared favorably with the
most reactive high-valent metal-oxidants, and showcases the exceptional
reactivity of late transition metalâoxygen adducts
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