scholarly journals Chemical cross-linking analyses of ox neurofilaments

1986 ◽  
Vol 234 (3) ◽  
pp. 587-591 ◽  
Author(s):  
M J Carden ◽  
P A M Eagles
Keyword(s):  
Ionic Strength ◽  
Tryptic Digestion ◽  
Cross Linking ◽  
Cross Link ◽  
Close Proximity ◽  
Neurofilament Polypeptides ◽  
Phenanthroline Complex ◽  
Low Ionic Strength ◽  
Polypeptide Chains ◽  
Chemical Cross Linking

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among ‘rod-domain’ fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.

scholarly journals The study of cell-death proteins in the outer mitochondrial membrane by chemical cross-linking

1997 ◽  
Vol 325 (2) ◽  
pp. 321-324 ◽  
Author(s):  
Sohail T. ALI ◽  
John R. COGGINS ◽  
Howard T. JACOBS
Keyword(s):  
Cell Death ◽  
Mitochondrial Membrane ◽  
Mitochondrial Protein ◽  
Surface Proteins ◽  
Cross Linking ◽  
Outer Mitochondrial Membrane ◽  
Cross Link ◽  
Cell Death Protein ◽  
Chemical Cross Linking

Chemical cross-linking was used to study the interactions of the anti-cell-death protein Bcl2 with other proteins in the outer mitochondrial membrane. Cross-linking of mitochondrial surface proteins produced a large Bcl2-containing complex (> 200 kDa), and a Bcl2-derived peptide was shown to cross-link specifically with a mitochondrial protein identified by immunoblotting as Raf-1 kinase.

scholarly journals Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa Rebers ◽  
Raffael Reichsöllner ◽  
Sophia Regett ◽  
Günter E. M. Tovar ◽  
Kirsten Borchers ◽  
...  
Keyword(s):  
Near Infrared ◽  
Cross Linking ◽  
Cross Link ◽  
Scanning Calorimetry ◽  
Link Density ◽  
Double Bond Conversion ◽  
Cross Links ◽  
Physical And Chemical ◽  
The Impact ◽  
Chemical Cross Linking

AbstractGelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

“Out-Gel” Tryptic Digestion Procedure for Chemical Cross-Linking Studies with Mass Spectrometric Detection

2014 ◽  
Vol 13 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Evgeniy V. Petrotchenko ◽  
Jason J. Serpa ◽  
Ashley N. Cabecinha ◽  
Mary Lesperance ◽  
Christoph H. Borchers
Keyword(s):  
Mass Spectrometric ◽  
Mass Spectrometric Detection ◽  
Tryptic Digestion ◽  
Cross Linking ◽  
Digestion Procedure ◽  
Chemical Cross Linking

7-Diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin, a fluorescent probe of the hydrophobic cleft in the tropomyosin coiled coil

1988 ◽  
Vol 66 (8) ◽  
pp. 1805-1808 ◽  
Author(s):  
Leslie D. Burtnick ◽  
Anita Racic
Keyword(s):  
Ionic Strength ◽  
Skeletal Muscles ◽  
Coiled Coil ◽  
Cysteine Residue ◽  
Deoxyribonuclease I ◽  
Low Ionic Strength ◽  
Hydrophobic Cleft ◽  
Polypeptide Chains ◽  
High Degree ◽  
Rabbit Skeletal Muscles

A sulfhydryl-specific fluorescent reagent, 7-diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin (DCIA) was used to label cysteine residues on tropomyosin (TM) from rabbit cardiac and rabbit skeletal muscles. The emission maximum at 486 nm, the high degree of fluorescence polarization, and the limited accessibility of the bound probe to quenching by iodide suggest that the probe is bound in the hydrophobic cleft between polypeptide chains of the TM coiled coil, as well as being bound covalently at a cysteine residue. The labelled TMs retain their abilities to bind F-actin and are able to interact with deoxyribonuclease I. They, however, show a reduced tendency to aggregate into filaments in low ionic strength solutions.

scholarly journals Crosstalk between CST and RPA regulates RAD51 activity during replication stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kai-Hang Lei ◽  
Han-Lin Yang ◽  
Hao-Yen Chang ◽  
Hsin-Yi Yeh ◽  
Dinh Duc Nguyen ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Replication Fork ◽  
Protein A ◽  
Replication Stress ◽  
High Ionic Strength ◽  
Close Proximity ◽  
Fork Stalling ◽  
Low Ionic Strength ◽  
Exchange Activity

AbstractReplication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

scholarly journals Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

1978 ◽  
Vol 77 (2) ◽  
pp. 488-506 ◽  
Author(s):  
G Kreibich ◽  
CM Freienstein ◽  
BN Pereyra ◽  
BL Ulrich ◽  
DD Sabatini
Keyword(s):  
Binding Sites ◽  
Cross Linking ◽  
Ribosomal Subunits ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Close Proximity ◽  
Microsomal Membranes ◽  
Cross Links ◽  
Polypeptide Chains ◽  
Specific Membrane

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.

scholarly journals Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

1999 ◽  
Vol 19 (5) ◽  
pp. 3788-3797 ◽  
Author(s):  
Quinn M. Eastman ◽  
Isabelle J. Villey ◽  
David G. Schatz
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Cross Linking ◽  
Specific Interactions ◽  
Cross Link ◽  
Site Specific ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Close Proximity ◽  
Rag Proteins

ABSTRACT V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.

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