13C-detected protonless NMR spectroscopy of proteins in solution

Bermel, Wolfgang; Bertini, Ivano; Felli, Isabella C.; Piccioli, Mario; Pierattelli, Roberta

doi:10.1016/j.pnmrs.2005.09.002

Cited by 221 publications

(243 citation statements)

References 96 publications

(194 reference statements)

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“…Standard 1 H-detected triple-resonance NMR experiments for backbone resonance assignment were recorded on 0.5-to 1-mM 13 C, 15 N-labeled samples at 298 K ([2Fe-2S]-anamorsin and [2Fe-2S]-CIAPIN1-single) and at 308 K (FMNNdor1, 1-174 aa), using a Bruker AVANCE 500 MHz spectrometer. 13 C-detected protonless NMR experiments (26) [CBCACO-in phase antiphase (IPAP), CACO-IPAP, CON-IPAP, CBCACON-IPAP, and CBCANCO-IPAP] acquired on a Bruker AVANCE 700 spectrometer, equipped with a cryogenically cooled probehead optimized for 13 C-direct detection, were also used for sequence-specific resonance assignment (N, C′, Cα, and Cβ) of [2Fe-2S]-anamorsin and [2Fe-2S]-CIA-PIN1-single. 13 C-direct-detected NMR experiments (26) acquired on 13 C, 15 Nlabeled [2Fe-2S]-anamorsin allowed us to obtain the backbone resonance assignment of all linker residues, with the exception of three (173-175).…”

Section: Methodsmentioning

confidence: 99%

“…When the anamorsin/FMN-Ndor1 interaction was monitored on a 13 C, 15 Nlabeled oxidized [2Fe-2S]-anamorsin sample by 13 C-direct detection CON, CACO, and CBCACO NMR experiments (Fig. S5), which experience increased spectral resolution for unstructured proteins (26), the spectral changes are located in the region preceding the CX 8 CX 2 CXC motif (residues 185-223), encompassing the last 38 residues of the linker. This region can be partitioned in two main areas characterized by specific amino acid content: a highly hydrophobic area (residues 188-202) and a negatively charged area (residues 204-223) (Fig.…”

Section: Structural Characterization Of the C-terminal Region Of [2fementioning

confidence: 99%

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Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Banci

Bertini

Calderone

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Biogenesis of iron-sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron-sulfur protein assembly machinery, two human key proteins-NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsinform a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron-sulfur cluster proteins. The Ndor1-anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.Fe/S protein maturation | CIAPIN1 domain | diflavin reductase I ron-sulfur clusters (ISCs) are ancient inorganic cofactors that are crucial for many protein functions in eukaryotic and bacterial cells (1-3). The clusters are composed of inorganic sulfide and ferric/ ferrous iron atoms, the latter being preferentially coordinated by cysteinyl residues (4-6). Because inorganic sulfide and ferrous/ferric iron atoms are toxic in vivo, biosynthesis of ISC proteins within cells is a highly regulated process that requires complex protein machineries for the mobilization of Fe and S atoms from appropriate sources, for their assembly into ISC forms and their final delivery to the recipient proteins (7-9). Three distinct protein machineries are operative and essential in the (nonplant) eukaryotic cells for the biogenesis of ISC proteins: (i) the ISC assembly machinery in the mitochondrial matrix, (ii) the ISC export machinery located in the mitochondrial intermembrane space, and (iii) the cytosolic iron-sulfur protein assembly (CIA) machinery.The CIA machinery comprises several proteins (10, 11), among which one named Dre2 has been recently identified in yeast (12). The C-terminal domain of Dre2 (residues 228-348) is able to bind two ISCs, a [2Fe-2S] and a [4Fe-4S] (12, 13). The [2Fe-2S] cluster of Dre2 receives electrons from a cytosolic diflavin reductase, termed Tah18 (13), which contains a FAD and a FMN prosthetic group, respectively, bound in two distinct structural domains, to accept electrons from NADPH (14). Dre2 and Tah18 are protein partners forming a stable complex in vivo (13, 15). The C-terminal region of Dre2 (residues 173-348) is fundamenta...

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Section: Methodsmentioning

confidence: 99%

Section: Structural Characterization Of the C-terminal Region Of [2fementioning

confidence: 99%

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Banci

Bertini

Calderone

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The 1 H spin systems of the aromatic rings of His residues were identified using a 2D 1 H-15 N HSQC type spectrum with a INEPT constant optimised for 2 J detection. 2D 13 C-detected CON, CACO, CBCACO and COSY (Bermel et al 2006) were performed to extend the assignment of backbone C/N and Cb resonances of residues which NH were not detected in the [ 15 N, 1 H]-HSQC, and to confirm the assignments obtained with standard triple-resonance experiments.…”

Section: Methods and Experimentsmentioning

confidence: 99%

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

et al. 2007

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We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13 C-detection experiments. Biological contextAn orange-coloured protein (ORP), 117 residues long, was isolated from the sulphate reducing bacterium Desulfovibrio gigas grown in a lactate-sulphate medium under anaerobic conditions (Bursakov et al. 2004). Sequence homology search through sequenced or partially sequenced genomes shows that this gene is present in organisms that are synthrophic with methanogenic archaea, such as other species of Desulfovibrio (D.), as D. vulgaris Hildenborough and D. desulfuricans G20. However, the sequence identity between D. gigas ORP and the corresponding ORF in these organisms is 48% and 42%, respectively and no structural information is available for any of them. Moreover, a recent study using D. vulgaris Hildenborough and Methanosarcina barkeri has showed that the gene that codes for this protein is one of three putative encoding-genes whose expression is increased when the growth conditions are switched from syntrophic to sulphate reducing (Scholten et al. 2007), making the study of this protein more imperative in order to understand its physiological role.To our knowledge, the D. gigas ORP was the first of this family of proteins to be isolated in the holo-form. The metal cluster is composed of two molybdenum and one copper ions arranged in a novel mixed-metal sulphide cluster of the type [S 2 MoS 2 CuS 2 MoS 2 ] 3-, which is noncovalently bound to the polypeptide chain (Bursakov et al. 2004, George et al. 2000. However, the ORP expressed heterologously in Escherichia. coli does not contain the metal cluster, but attempts are currently being made in order to reconstitute the protein with a metal cluster.Solution structural studies have been performed in two other proteins of this family, the homologue from Thermotoga maritima (TM1290) and the one from Methanobacterium thermoautotrophicum (MTH1175) (Cort et al. 2000, Etezady-Esfarjani et al. 2004). Both proteins were obtained through the structural genomic approach: the ORFs with no known function were chosen from the complete genome of these bacteria, heterologously expressed and their structure determined in order to obtain functional insights. However, besides presenting low sequence identity with D. gigas ORP, 35% (TM1290) and 32% (MTH1175), no function has been identified so far for these proteins. Attending to the fact that these homology values are in the limit to apply homology modelling, we started our studies by the resonance assignment in order to obtain preliminary structural information about the apo-ORP from D. gigas. This is the starting point to further pursue the studies using the reconstituted holo-protein, in order to determine the binding site of the metal cluster and to obtain further structure-function insights on ...

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“…Die T 1 -Relaxationszeiten für Protonen (nichtselektiv aufgenommen) wurden aus Serien von HSQC-Spektren bestimmt. Die T 1 -Relaxationszeiten für 13 C-Kerne erhielten wir aus CaCO-und CON-Experimenten, [7,8] wobei wir eine "Inversion-recovery"-Einheit zu Beginn der Pulssequenz verwendeten. Die PRE-Strukturen von Ubiquitin und MBP wurden in der PDB-Datenbank unter den Zugangsnummern 2klg (Ubiquitin) und 2klf (MBP) hinterlegt.…”

Section: Experimentellesunclassified

“…Durch Entwicklungen auf dem Gebiet der Methoden (z. B. transverse relaxation-optimized spectroscopy (TROSY [1] ), dipolare Restkopplungen (RDCs [2,3] ), Deuterierung, [4] stereoarray isotope labeling (SAIL [5] ), Methyl-TROSY [6] und direkte 13 C-Detektion [7][8][9] ) konnte das Größenlimit für NMRspektroskopisch in Lösung analysierbare Proteine in den letzten 10 Jahren angehoben werden. Jedoch ist die Zahl an Strukturen für Proteine über 30 kDa, die NMR-spektroskopisch gelöst wurden, noch immer recht gering.…”

unclassified

Der Einsatz von Relaxationserhöhungen in einer paramagnetischen Umgebung zur Proteinstrukturbestimmung mit NMR‐Spektroskopie

Bermel

Zangger

2009

Angewandte Chemie

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Ein Mehr an Informationen: Relaxationserhöhungen durch eine inerte paramagnetische Umgebung (PREs) wurden zusammen mit einem eingegrenzten NOE‐Datensatz in einem modellfreien Strukturbestimmungsverfahren genutzt. Strukturen für zwei Proteine – Ubiquitin (8 kDa) und das Maltose‐bindende Protein (42 kDa; im Komplex mit β‐Cyclodextrin) – wurden mithilfe von PREs und NOEs zwischen austauschenden Protonen ermittelt.

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13C-detected protonless NMR spectroscopy of proteins in solution

Cited by 221 publications

References 96 publications

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Der Einsatz von Relaxationserhöhungen in einer paramagnetischen Umgebung zur Proteinstrukturbestimmung mit NMR‐Spektroskopie

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