scholarly journals Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

1988 ◽  
Vol 256 (2) ◽  
pp. 521-528 ◽  
Author(s):  
S D Patel ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Membrane Lipid ◽  
Protein Domain ◽  
Cross Linking ◽  
Hydrophobic Domain ◽  
Iron Protein ◽  
Dimethyl Suberimidate ◽  
Cross Links ◽  
Monospecific Antisera ◽  
Chemical Cross Linking

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.

scholarly journals Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart

1985 ◽  
Vol 227 (2) ◽  
pp. 467-474 ◽  
Author(s):  
M W J Cleeter ◽  
S H Banister ◽  
C I Ragan
Keyword(s):  
Ethylene Glycol ◽  
Active Site ◽  
Protein Fraction ◽  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Iron Protein ◽  
Bovine Heart ◽  
Cross Links ◽  
Hydrophobic Shell ◽  
Chemical Cross Linking

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.

scholarly journals Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

1988 ◽  
Vol 256 (2) ◽  
pp. 529-535 ◽  
Author(s):  
S D Patel ◽  
M W J Cleeter ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Submitochondrial Particles ◽  
Mitochondrial Inner Membrane ◽  
Cytoplasmic Face ◽  
The Matrix ◽  
Dimethyl Suberimidate ◽  
Membrane Reaction ◽  
Cytoplasmic Side ◽  
Chemical Cross Linking

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

1977 ◽  
Vol 55 (1) ◽  
pp. 43-49 ◽  
Author(s):  
M. Kapoor ◽  
M. D. O'Brien
Keyword(s):  
Pyruvate Kinase ◽  
Functional Groups ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Complex Cross ◽  
Dimethyl Suberimidate ◽  
Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

Abstract 263: Isotopic Labeling and Chemical Cross-Linking Reveals Intermolecular Contacts and Dynamics of Apolipoprotein A-I Structure in Reconstituted HDL Particles

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamie Morris ◽  
Martin K Jones ◽  
Gang Ren ◽  
Jere Segrest ◽  
W Sean Davidson
Keyword(s):  
Conformational Dynamics ◽  
Density Lipoprotein ◽  
Isotopic Labeling ◽  
Peptide Fragmentation ◽  
Cross Linking ◽  
Naturally Occurring ◽  
Intermolecular Contacts ◽  
Cross Links ◽  
Mass Shifts ◽  
Chemical Cross Linking

Apolipoprotein (apo)A-I has been proposed to adopt a number of different, but related, structures when in contact with lipid. The technique of chemical cross-linking has recently been used to determine the spatial relationships between two molecules of apoA-I in reconstituted high density lipoprotein (rHDL) particles. However, this technique is limited in that it cannot unequivocally distinguish between intramolecular and intermolecular contacts. To address this issue, we have produced two forms of recombinant apoA-I that contain the naturally occurring isotope of nitrogen (N14) or a stable isotope (N15). These forms were mixed 1:1 and then used to produce reconstituted HDL particles with synthetic lipids. The resulting mass shifts (readily detectable in the mass spectrometer) were exploited to unambiguously distinguish between intramolecular (N14 to N14 or N15 to N15) and intermolecular (N14 to N15) cross-linked peptides. An additional benefit of this approach was the ability to identify cross-links with high certainty without the need for peptide fragmentation, allowing for dramatic increases in method sensitivity. We studied highly homogeneous rHDL particles made with the fully saturated phospholipid palmitoyl steroyl phosphatidylcholine (PSPC) to minimize apoA-I conformational dynamics. These particles were 98Å in diameter, contained two molecules of apoA-I, approximately 155 molecules of PC, and were discoidal in shape by cryo EM. We identified 30 cross-links (17 intramolecular, 13 intermolecular) that were overall consistent with the double belt model in which both apoA-I molecules wrap around a bilayer of lipids in an antiparallel orientation. Unambiguous intramolecular linkages between the N- and C-terminal regions of apoA-I were inconsistent with the ‘double super helix’ variant of the belt model. We identified several low abundance cross-links that suggest that the N-terminus may be conformationally dynamic and may spend some time folded back across the molecule as proposed for the ‘belt and buckle’ belt model.

Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

1986 ◽  
Vol 64 (3) ◽  
pp. 229-237
Author(s):  
Nobuhito Sone ◽  
Cynthia Hou ◽  
Philip D. Bragg
Keyword(s):  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Thermophilic Bacterium ◽  
Cross Linking ◽  
Dimethyl Suberimidate ◽  
The Cross ◽  
Third Dimension ◽  
Chemical Cross Linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

Calcium Ion Binding Within Fibrinogen Fragment D

1981 ◽  
Author(s):  
David W Britton ◽  
Jan S Lawrie ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Cross Linking ◽  
C Terminus ◽  
Dimethyl Suberimidate ◽  
High Concentrations ◽  
The Stability ◽  
Considerable Body ◽  
Chemical Cross Linking ◽  
Compact Conformation

Fibrinogen contains a number of strongly bound calcium. ions and a considerable body of evidence new exists to show that the plasmin degradation product fragment D contains one strongly bound calciumion. It is also established that this calcium ion has a notable effect on the plasmin resistance of the molecule. Previous work from this laboratory strongly suggests that the binding site is located towards the C-terminus of the γ chain. We have also investigated the influence of calcium. ions on the conformation of fragment D by ultracentrifugation and chemical cross-linking. In the presence of calcium ions there is a preponderance of intra-molecular cross-linking even at high concentrations of fragment D using bisimidates such as dimethyl suberimidate and dimethyl adipimidate. In the absence of calcium. ions there is an increase in the extent of inter-molecular cross-linking. From such evidence we would propose that calcium ions stabilise a compact conformation within fragment D. The presence of calcium ions also affects the stability of the D:E complex.

scholarly journals High-density chemical cross-linking for modeling protein interactions

2019 ◽  
Vol 117 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Julian Mintseris ◽  
Steven P. Gygi
Keyword(s):  
Mass Spectrometry ◽  
Method Development ◽  
Conformational Dynamics ◽  
Complex Function ◽  
Computational Cost ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Proteasome Subunits ◽  
Cross Links ◽  
Chemical Cross Linking

Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are self-consistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique cross-linked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.

scholarly journals A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

1984 ◽  
Vol 99 (1) ◽  
pp. 188-198 ◽  
Author(s):  
N G Kravit ◽  
C S Regula ◽  
R D Berlin
Keyword(s):  
Molecular Weight ◽  
Room Temperature ◽  
Molecular Form ◽  
Cross Linking ◽  
Electrophoretic Patterns ◽  
Minority Species ◽  
Associated Proteins ◽  
Dimethyl Suberimidate ◽  
Quantitative Examination ◽  
Chemical Cross Linking

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

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