scholarly journals Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Jason C Crack ◽  
Melissa Y Y Stewart ◽  
Nick E Le Brun
Keyword(s):  
Mass Spectrometry ◽  
Structure Function ◽  
Degradation Products ◽  
Spectroscopic Techniques ◽  
Cluster Assembly ◽  
Average Increase ◽  
Cysteine Desulfurase ◽  
Sulphide Ions ◽  
Unambiguous Assignment

Abstract The ability to specifically label the sulphide ions of protein-bound iron–sulphur (FeS) clusters with 34S isotope greatly facilitates structure–function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the O-acetyl-l-serine sulfhydrylase CysK to generate 34S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply 34S2− for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.

scholarly journals Iron–sulfur cluster biosynthesis

2008 ◽  
Vol 36 (6) ◽  
pp. 1112-1119 ◽  
Author(s):  
Sibali Bandyopadhyay ◽  
Kala Chandramouli ◽  
Michael K. Johnson
Keyword(s):  
Scaffold Proteins ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Eukaryotic Organisms ◽  
Almost All ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

Using Mass Spectrometry to Investigate the Structural Features of Photocrosslinked Co-Networks based on Gelatin and Poly(ethylene glycol) Methacrylates

2012 ◽  
Vol 1403 ◽  
Author(s):  
Benjamin F. Pierce ◽  
Axel T. Neffe ◽  
Andreas Lendlein
Keyword(s):  
Mass Spectrometry ◽  
Ethylene Glycol ◽  
Degradation Products ◽  
Structural Features ◽  
Valuable Insight ◽  
Poly Ethylene Glycol ◽  
Buffer Solution ◽  
Molecular Weight Range ◽  
Poly Ethylene

ABSTRACTGelatin was functionalized with glycidyl methacrylate and photocrosslinked in the presence of poly(ethylene glycol) dimethacrylate (PEGDMA) or poly(ethylene glycol) monomethacrylate (PEGMA) to create a biopolymer-based system with tailorable properties. These co-networks were hydrolyzed using 6 M HCl and the degradation products were analyzed and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. This technique successfully identified gelatin-derived peptides such as FLPEPPE, SFLPEPPE, and SFLPEPPEE as well as an accompanying PEG-g-poly(methacrylic acid) component. No oligo- or polymethacrylates were monitored at any molecular weight range above m/z = 500, which indicated that they possessed lower molecular weights. An in vitro hydrolytic degradation experiment performed in pH 7.4 PBS buffer solution at 37 °C showed that these networks, which were prepared without the addition of a potentially toxic photoinitiator, exhibited mass loss of up to 50 wt% at 6 weeks of incubation time. These results provide valuable insight into how these functional gelatin-based co-network biomaterials will perform in a biological setting.

Biogenesis and Transfer of Iron-Sulfur Clusters from Acidithiobacillus ferrooxidans

2013 ◽  
Vol 825 ◽  
pp. 198-201 ◽  
Author(s):  
Jian She Liu ◽  
Lin Qian ◽  
Chun Li Zheng
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Binding Protein ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins

Iron-sulfur (Fe-S) proteins are ubiquitous and participate in multiple essential functions of life. However, little is currently known about the mechanisms of iron-sulfur biosynthesis and transfer in acidophilic microorganisms. In this study, the IscS, IscU and IscA proteins from Acidithiobacillus ferrooxidans were successfully expressed in Escherichia coli and purified by affinity chromatography. The IscS was a cysteine desulfurase which catalyzes desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly. Purified IscU did not have an iron-sulfur cluster but could act as a scaffold protein to assemble the [2Fe-2S] cluster in vitro. The IscA was a [4Fe-4S] cluster binding protein, but it also acted as an iron binding protein. Further studies indicated that the iron sulfur clusters could be transferred from pre-assembled scaffold proteins to apo-form iron sulfur proteins, the reconstituted iron sulfur proteins could restore their physiological activities.

scholarly journals Conformational stability of six truncated cHMG1a proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry.

2001 ◽  
Vol 48 (4) ◽  
pp. 1131-1136 ◽  
Author(s):  
I Petry ◽  
J R Wigniewski ◽  
Z Szewczuk
Keyword(s):  
Mass Spectrometry ◽  
Degradation Products ◽  
Conformational Stability ◽  
Biological Significance ◽  
High Mobility ◽  
Intact Protein ◽  
Ionization Mass ◽  
Hmg Proteins ◽  
Hmg Protein

The high mobility group (HMG) proteins are abundant non-histone components of eukaryotic chromatin. The presence of C-terminal acidic tails is a common feature of the majority of HMG proteins. Although the biological significance of the acidic domains is not clear, they are conferring conformational and metabolic stability to the proteins in vitro. Moreover, the length and net charge of the acidic tails affect the strength of HMG protein interaction with DNA. Synthesis of an insect HMG protein by standard recombinant technology in bacteria leads to a mixture of the intact protein (cHMG1a-(1-113) (I)) and a series of its degradation products truncated at the C tail: cHMG1a-(1-111) (II); cHMG1a-(1-110) (III); cHuMGla-(1-109) (IV); cHMG1a-(1-108) (V); cHMG1a-(1-107) (VI); cHMG1a-(1-106) (VII). The proteins differ from each other only by the number of amino-acid residues at the C-terminal tail. We used H/D exchange mass spectrometry to characterize the stability of the proteins directly in their mixture. The results show that the proteins I-V and VII have very similar conformations. The protein VI is less compact and exchanges its protons faster than the others. It may be concluded that the C-terminal tail influences the conformation of the cHMG1a protein and that individual residues in this part of the protein play a key role in its compactness.

scholarly journals Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity

2018 ◽  
Author(s):  
Nicholas G. Fox ◽  
Alain Martelli ◽  
Joseph F. Nabhan ◽  
Jay Janz ◽  
Oktawia Borkowska ◽  
...  
Keyword(s):  
Protein Complex ◽  
De Novo ◽  
Cluster Assembly ◽  
Wild Type ◽  
Complex Activity ◽  
Cysteine Desulfurase ◽  
Iron Sulfur ◽  
Ethylenediaminetetracetic Acid ◽  
Piperazineethanesulfonic Acid

ABSTRACTThe human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140 L, and M140 V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU.ABBREVIATIONSACPacyl carrier transfer proteinBLIbiolayer interferometryBSAbovine serum albuminCDcircular dichroismDMPDNN-dimethyl-p-phenylenediamineDSFdifferential scanning fluorimetryDTTdithiothreitol; EDTA, ethylenediaminetetracetic acidFe-Siron sulfurFRDAFriedreich’s ataxiaFXNfrataxinHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidIPTGisopropyl β-D-1-thiogalactopyranosidePLPpyridoxal 5′-phosphateSDprotein complex composed of NFS 1 and ISD11SDUprotein complex composed of NFS 1, ISD11ISCUSDUF, protein complex composed of NFS 1, ISD11, ISCU, and frataxinTCAtrichloroacetic acidTCEPtris(2-carboxyethyl) phosphineTristris(hydroxymethyl)aminomethane

scholarly journals Protein Glycation in Diabetes as Determined by Mass Spectrometry

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Annunziata Lapolla ◽  
Laura Molin ◽  
Pietro Traldi
Keyword(s):  
Mass Spectrometry ◽  
Degradation Products ◽  
Glycated Haemoglobin ◽  
Maldi Mass Spectrometry ◽  
Enzymatic Digestion ◽  
Protein Glycation ◽  
Chronic Complications ◽  
Glycation End Products

Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). The preliminary studies carried out byin vitroprotein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA) and immunoglobulin G (IgG). Enzymatic degradation products ofin vitroglycated HSA were studied in order to simulate thein vivoenzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs) peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

scholarly journals Mechanism of Iron–Sulfur Cluster Assembly: In the Intimacy of Iron and Sulfur Encounter

Inorganics ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 55
Author(s):  
Batoul Srour ◽  
Sylvain Gervason ◽  
Beata Monfort ◽  
Benoit D’Autréaux
Keyword(s):  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Physiological Relevance ◽  
Client Proteins ◽  
Sulfide Ions ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are protein cofactors of a multitude of enzymes performing essential biological functions. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe–S clusters are assembled and a cysteine desulfurase that provides sulfur in the form of a persulfide. The sulfide ions are produced by reductive cleavage of the persulfide, which involves specific reductase systems. Several other components are required for Fe–S biosynthesis, including frataxin, a key protein of controversial function and accessory components for insertion of Fe–S clusters in client proteins. Fe–S cluster biosynthesis is thought to rely on concerted and carefully orchestrated processes. However, the elucidation of the mechanisms of their assembly has remained a challenging task due to the biochemical versatility of iron and sulfur and the relative instability of Fe–S clusters. Nonetheless, significant progresses have been achieved in the past years, using biochemical, spectroscopic and structural approaches with reconstituted system in vitro. In this paper, we review the most recent advances on the mechanism of assembly for the founding member of the Fe–S cluster family, the [2Fe2S] cluster that is the building block of all other Fe–S clusters. The aim is to provide a survey of the mechanisms of iron and sulfur insertion in the scaffold proteins by examining how these processes are coordinated, how sulfide is produced and how the dinuclear [2Fe2S] cluster is formed, keeping in mind the question of the physiological relevance of the reconstituted systems. We also cover the latest outcomes on the functional role of the controversial frataxin protein in Fe–S cluster biosynthesis.

scholarly journals High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4071
Author(s):  
Elisa Maffioli ◽  
Zhenze Jiang ◽  
Simona Nonnis ◽  
Armando Negri ◽  
Valentina Romeo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Proteolytic Activity ◽  
High Resolution Mass Spectrometry ◽  
Degradation Products ◽  
Quantitative Information ◽  
Peptidase Activity ◽  
Vitro Assay ◽  
Unbiased Manner

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 310-315 ◽  
Author(s):  
P W Koppert ◽  
E Hoegee-de Nobel ◽  
W Nieuwenhuizen
Keyword(s):  
Enzyme Immunoassay ◽  
Degradation Products ◽  
Blood Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Strong Positive Reaction ◽  
Sandwich Type ◽  
Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

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