How Bonding in Manganous Phosphates Affects their Mn(II)-(31)P Hyperfine Interactions.

International audienceManganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn(II) electron-nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn(II)-(31)P hyperfine interactions of various Mn(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese-phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ (31)P ENDOR spectra of cellular Mn(II) complexes

Chloroplastic Hsp100 chaperones ClpC2 and ClpD interact <it>in vitro</it> with a transit peptide only when it is located at the N-terminus of a protein

<p>Abstract</p> <p>Background</p> <p>Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.</p> <p>Results</p> <p>We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from <it>Arabidopsis thaliana</it> (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione <it>S</it>-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.</p> <p>Conclusions</p> <p>AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.</p

How Bonding in Manganous Phosphates Affects their Mn(II)–³¹P Hyperfine Interactions

Manganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn­(II) electron–nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn­(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn­(II)–³¹P hyperfine interactions of various Mn­(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese–phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn­(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ ³¹P ENDOR spectra of cellular Mn­(II) complexes

Pulse Electron Double Resonance Detected Multinuclear NMR Spectra of Distant and Low Sensitivity Nuclei and Its Application to the Structure of Mn(II) Centers in Organisms.

International audienceThe ability to characterize the structure of metal centers beyond their primary ligands is important to understanding their chemistry. High-magnetic-field pulsed electron double resonance detected NMR (ELDOR-NMR) is shown to be a very sensitive approach to measuring the multinuclear NMR spectra of the nuclei surrounding Mn(II) ions. Resolved spectra of intact organisms with resonances arising from (55)Mn, (31)P, (1)H, (39)K, (35)Cl, (23)Na, and (14)N nuclei surrounding Mn(2+) centers were obtained. Naturally abundant cellular (13)C could be routinely measured as well. The amplitudes of the (14)N and (2)H ELDOR-NMR spectra were found to be linearly dependent on the number of nuclei in the ligand sphere. The evolution of the Mn(II) ELDOR-NMR spectra as a function of excitation time was found to be best described by a saturation phenomenon rather than a coherently driven process. Mn(II) ELDOR-NMR revealed details about not only the immediate ligands to the Mn(II) ions but also more distant nuclei, providing a view of their extended structures. This will be important for understanding the speciation and chemistry of the manganese complexes as well as other metals found in organisms

An environmental scenario for the earliest hominins in the Iberian Peninsula: Early Pleistocene palaeovegetation and palaeoclimate

International audienceThe Early Pleistocene deposits of the Iberian Peninsula provided some of the oldest hominin fossil sites of Western Europe. Evidence also shows that early Homo thrived in the Mediterranean peninsulas during the Early Pleistocene 'interglacial' phases. To assess the role of climatic conditions on early human environments, the present work features a quantitative palaeoclimatic analysis for a number of Early Pleistocene macroflora and pollen assemblages located at different geographical locations where hominin activity has been recorded. The results picture a cyclic climate with a possible latitudinal aridity gradient. Warm and humid ('interglacial') phases would have been wetter and slightly warmer than the modern climate. During cooler and drier ('glacial') phases, temperature and precipitation were comparatively milder and more similar to modern ones. The favourable conditions during the humid phases may have allowed for the earliest hominin communities arriving in Europe to rapidly thrive during 'interglacial' periods. The present climatic quantification suggests that hominins in Iberia may have survived these mild 'glacial' Early Pleistocene stages

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins

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3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

International audienceGlutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs 180) contain long N-and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs 180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH 180 (mL-GDH 180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH 180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs