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1.
J Magn Reson ; 364: 107708, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38901173

RESUMEN

Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13C13C and heteronuclear 1H15N, 1H13C and 15N13C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3-4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples.

2.
Nat Commun ; 14(1): 6706, 2023 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-37872144

RESUMEN

Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently.


Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Peptidoglicano/química , Hidrolasas , Lisostafina/análisis , Lisostafina/química , Espectrometría de Masas/métodos , Pared Celular/química
3.
J Am Chem Soc ; 145(19): 10700-10711, 2023 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-37140345

RESUMEN

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive µs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.


Asunto(s)
Peróxido de Hidrógeno , Peroxirredoxinas , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Oxidación-Reducción , Peróxido de Hidrógeno/metabolismo , Conformación Molecular , Disulfuros/química
4.
Angew Chem Int Ed Engl ; 62(19): e202219314, 2023 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-36738230

RESUMEN

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-ß amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-ß architecture.


Asunto(s)
Péptidos beta-Amiloides , Amiloide , Resonancia Magnética Nuclear Biomolecular/métodos , Amiloide/química , Espectroscopía de Resonancia Magnética , Conformación Proteica , Péptidos beta-Amiloides/metabolismo
5.
J Hazard Mater ; 446: 130668, 2023 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-36608581

RESUMEN

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Uranio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Compuestos Férricos/metabolismo , Membrana Celular/metabolismo , Cationes/química , Cationes/metabolismo , Uranio/química , Proteínas de Unión al Calcio/metabolismo
6.
Methods Mol Biol ; 2199: 127-149, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33125648

RESUMEN

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.


Asunto(s)
Proteínas de Unión al ADN , Deuterio/química , Escherichia coli/química , Marcaje Isotópico , Proteínas Proto-Oncogénicas , Sistema Libre de Células/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dioxigenasas , Humanos , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
7.
Data Brief ; 31: 105865, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32642505

RESUMEN

Linker histone H1 plays a vital role in the packaging of DNA. H1 has a tripartite structure: a conserved central globular domain that adopts a winged-helix fold, flanked by highly variable and intrinsically unstructured N- and C-terminal domains. The datasets presented in this article include raw 2D and 3D BEST-TROSY NMR data [1H-15 N HSQC; 15 N and 13C HNCO, HN(CO)CACB, HNCACB, HN(CA)CO] recorded for NGH1x, a truncated version of H1x containing the N-terminal and globular domains, but lacking the C-terminal domain. Experiments were conducted on double-labelled (15 N and 13C) NGH1x in 'low' and 'high salt,' to investigate the secondary structure content of the N-terminal domain of H1x under these conditions. We provide modelled structures of NGH1x (in low and high salt) based on the assigned chemical shifts in PDB format. The high salt structure of NGH1x (globular domain of H1x [GH1x; PDB: 2LSO] with the H1x NTD) was docked to the nucleosome to generate NGH1x- and GH1x-chromatosomes. The GH1x-chromatosome was generated for comparative purposes to elucidate the role of the N-terminal domain. We present raw data trajectories of molecular dynamics simulations of these chromatosomes in this article. The MD dataset provides nanosecond resolution data on the dynamics of GH1x- vs NGH1x-chromatosomes, which is useful to elucidate the DNA binding properties of the N-terminal domain of H1x in chromatin, as well as the dynamic behaviour of linker DNA in these chromatosomes.

8.
Soft Matter ; 16(15): 3628-3641, 2020 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-32222755

RESUMEN

Dynamic covalent hydrogels crosslinked by boronate ester bonds are promising materials for biomedical applications. However, little is known about the impact of the crosslink structure on the mechanical behaviour of the resulting network. Herein, we provide a mechanistic study on boronate ester crosslinking upon mixing hyaluronic acid (HA) backbones modified, on the one hand, with two different arylboronic acids, and on the other hand, with three different saccharide units. Combining rheology, NMR and computational analysis, we demonstrate that carefully selecting the arylboronic-polyol couple allows for tuning the thermodynamics and molecular exchange kinetics of the boronate ester bond, thereby controlling the rheological properties of the gel. In particular, we report the formation of "strong" gels (i.e. featuring slow relaxation dynamics) through the formation of original complex structures (tridentate or bidentate complexes). These findings offer new prospects for the rational design of hydrogel scaffolds with tailored mechanical response.


Asunto(s)
Ácidos Borónicos/química , Ácido Hialurónico/química , Hidrogeles/química , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Reología
9.
Magn Reson (Gott) ; 1(2): 331-345, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-37904819

RESUMEN

We introduce ssNMRlib, a comprehensive suite of pulse sequences and jython scripts for user-friendly solid-state nuclear magnetic resonance (NMR) data acquisition, parameter optimization and storage on Bruker spectrometers. ssNMRlib allows the straightforward setup of even highly complex multi-dimensional solid-state NMR experiments with a few clicks from an intuitive graphical interface directly from the Bruker Topspin acquisition software. ssNMRlib allows the setup of experiments in a magnetic-field-independent manner and thus facilitates the workflow in a multi-spectrometer setting with a centralized library. Safety checks furthermore assist the user in experiment setup. Currently hosting more than 140 1D to 4D experiments, primarily for biomolecular solid-state NMR, the library can be easily customized and new experiments are readily added as new templates. ssNMRlib is part of the previously introduced NMRlib library, which comprises many solution-NMR pulse sequences and macros.

10.
Biomol NMR Assign ; 13(1): 249-254, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30868366

RESUMEN

Human linker histone H1 plays a seminal role in eukaryotic DNA packaging. H1 has a tripartite structure consisting of a central, conserved globular domain, which adopts a winged-helix fold, flanked by two variable N- and C-terminal domains. Here we present the backbone resonance assignments of the N-terminal domain and globular domain of human linker histone H1x in the presence and absence of the secondary structure stabilizer sodium perchlorate. Analysis of chemical shift changes between the two conditions is consistent with induction of transient secondary structural elements in the N-terminal domain of H1x in high ionic strength, which suggests that the N-terminal domain adopts significant alpha-helical conformations in the presence of DNA.


Asunto(s)
Histonas/química , Resonancia Magnética Nuclear Biomolecular , Percloratos/farmacología , Compuestos de Sodio/farmacología , Humanos , Dominios Proteicos , Estructura Secundaria de Proteína
11.
J Magn Reson ; 300: 95-102, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30721859

RESUMEN

We propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural 13C abundance. The characteristic peak patterns detected in SOFAST-HMBC for each aromatic side chain allow straightforward assignment of all protons and carbons (including quaternary ones) of the aromatic ring, and for tyrosine and phenylalanine, connection to the CB of the aliphatic chain. The performance of SOFAST-HMBC is demonstrated for three small proteins (7-14 kDa) at millimolar sample concentration using modern high-field NMR instruments equipped with cryogenically cooled probes. Despite the low amount of NMR-active 13C nuclei in these samples, 1H-13C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h.


Asunto(s)
Carbono/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Aminoácidos/química , Isótopos de Carbono , Hidrocarburos Aromáticos , Muramidasa/química , Protones
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