Reagent For Cleavage Research Articles

Identification and Mapping of Protein−Protein Interactions by a Combination of Cross-Linking, Cleavage, and Proteomics

Protein-protein interactions are vital for almost all cellular functions, and many require the formation of multiprotein complexes. Identification of the macroscopic and microscopic protein interactions within these complexes is essential in understanding their mechanisms, both under physiologic as well as pathologic conditions. This review describes the current technology available to investigate interactions between proteins utilizing chemical cross-linking and site-directed cleavage reagents, outlining the necessary steps involved in identifying interacting proteins both in vitro and in vivo. Once interacting proteins are identified, more information about the architecture of the assemblies is necessary. Unique separation techniques coupled with cross-linking and mass spectrometry can now identify specific interaction sites and lead to the development of quaternary structural protein models. Furthermore, combination of these methods with proteomic approaches enables the identification and analysis of complex interactions in vivo. Finally, future directions in cross-linking methodologies are discussed.

A New Method for the Synthesis of Organotellurium Compounds by the Use of Reductive Cleavage of the Tellurium—Tellurium Bond with Lanthanum Metal.

It was confirmed that lanthanum metal is an efficient reagent for the reductive cleavage of the tellurium–tellurium bond of ditelluride. Alkyl phenyl tellurides were prepared by the reaction of dip...

A Mild and Selective Method for the Conversion of Oximes into Ketones and Aldehydes by the Use of N‐Bromophthalimide.

N-Bromophthalimide has been found to be an efficient and selective reagent for the mild oxidative cleavage of oximes to yield their corresponding carbonyl compounds in good to excellent yields.

A New Method for the Synthesis of Organotellurium Compounds by the Use of Reductive Cleavage of the Tellurium–Tellurium Bond with Lanthanum Metal

Abstract It was confirmed that lanthanum metal is an efficient reagent for the reductive cleavage of the tellurium–tellurium bond of ditelluride. Alkyl phenyl tellurides were prepared by the reaction of diphenyl ditelluride with primary and secondary alkyl halides in the presence of lanthanum metal and a catalytic amount of iodine in moderate to good yields. The reaction was accelerated by the addition of HMPA. In addition, the reduction of ditelluride with lanthanum metal, followed by the addition of acyl or aroyl chlorides or α,β-unsaturated ester, provided the Te-phenyl tellurol esters and the β-phenyl telluro ester in moderate yields.

Imidazolium Dichromate Adsorbed on Alumina: A Convenient and Inexpensive Reagent for the Cleavage of C=N Functionalities

Imidazolium Dichromate Adsorbed on Alumina: A Convenient and Inexpensive Reagent for the Cleavage of C=N Functionalities

Green Traceless Cleavage from Resin-Bound Selenium and Tellurium and Analysis by 2D29Si/1H HR-MAS NMR Spectroscopy

A novel protocol for traceless cleavage from resin-bound selenium and tellurium is described. The toxic tributyltin hydride that has been used previously as reagent for cleavage from these resins by homolysis has been replaced by the less-toxic tris(trimethylsilyl)silane and tris(trimethylsilyl)germane. In addition, the remaining resin-bound chalcogenyl silane and germane residues were characterised by the use of high-resolution magic-angle spinning (HR-MAS) 2D 29Si/1H NMR spectroscopy.

Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities

Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.

Microwave‐Promoted Selective Regeneration of Carbonyl Compounds from Oximes Using N‐Bromosaccharin.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Identifying protein interactions by hydroxyl-radical protein footprinting.

Hydroxyl-radical protein footprinting is a straightforward and direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that shows regions of the protein that change their susceptibility to cleavage while bound to a ligand.

Allyl, Methallyl, Prenyl, and Methylprenyl Ethers as Protected Alcohols: Their Selective Cleavage with Diphenyldisulfone under Neutral Conditions.

AbstractFor Abstract see ChemInform Abstract in Full Text.

A Mild and Selective Method for the Conversion of Oximes into Ketones and Aldehydes by the Use of N-Bromophthalimide

N-Bromophthalimide has been found to be an efficient and selective reagent for the mild oxidative cleavage of oximes to yield their corresponding carbonyl compounds in good to excellent yields.

N,N‐Dibromobenzenesulfonamide as a Reagent for Oxidative Cleavage of Oximes to Their Parent Carbonyl Compounds under Non‐Aqueous and Aprotic Conditions.

Deprotection of different oximes to their parent aldehydes and ketones in high yields has been carried out by using N,N-dibromobenzenesulfonamide(dibromoamine-b) under mild conditions.

Microwave-Promoted Selective Regeneration of Carbonyl Compounds from Oximes Using N-Bromosaccharin

N-bromosaccharin was found to be an efficient reagent for the oxidative cleavage of oximes to the corresponding aldehydes and ketones under microwave irradiation in a domestic microwave oven.

Allyl, Methallyl, Prenyl, and Methylprenyl Ethers as Protected Alcohols: Their Selective Cleavage with Diphenyldisulfone under Neutral Conditions

Diphenyldisulfone is a mild and efficient reagent for selective cleavage of methylprenyl (2,3-dimethylbut-2-en-1-yl), prenyl (3-methylbut-2-en1-yl), and methallyl (2-methylallyl) ethers. These reaction conditions are compatible with the presence of other protecting groups such as acetals, acetates, and allyl, benzyl, and TBDMS ethers. Exposure of 2,3-dimethylbut-2-en-1-yl and 3-methylbut-2-en1-yl ethers to diphenyldisulfone led to the formation of 2,3-dimethylbuta-1,3-diene and isoprene, respectively. 2-Methylallyl ethers undergo isomerization to 2-methylpropenyl ethers, which are easily hydrolyzed into the corresponding free alcohols and isobutyraldehyde. [reaction: see text]

Quinolinium Fluorochromate as a Reagent for Selective Oxidative Cleavage of Oximes and Phenylhydrazones to Their Parent Carbonyl Compounds under Non‐Aqueous and Aprotic Conditions.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

A surface that is required for rapid formation of preinitiation complexes (PICs) was identified on the N-terminal domain (NTD) of the RNA Pol II general transcription factor TFIIA. Site-specific photocross-linkers and tethered protein cleavage reagents positioned on the NTD of TFIIA and assembled in PICs identified the SAGA subunit Spt8 and the TFIID subunit Taf4 as located near this surface. In agreement with these findings, mutations in Spt8 and the TFIIA NTD interact genetically. Using purified proteins, it was found that TFIIA and Spt8 do not stably bind to each other, but rather both compete for binding to TBP. Consistent with this competition, Spt8 inhibits the binding of SAGA to PICs in the absence of activator. In the presence of activator, Spt8 enhances transcription in vitro, and the positive function of the TFIIA NTD is largely mediated through Spt8. Our results suggest a mechanism for the previously observed positive and negative effects of Spt8 on transcription observed in vivo.

N,N-Dibromobenzenesulfonamide as a Reagent for Oxidative Cleavage of Oximes to Their Parent Carbonyl Compounds Under Non-Aqueous and Aprotic Conditions

Deprotection of different oximes to their parent aldehydes and ketones in high yields has been carried out by using N,N-dibromobenzenesulfonamide(dibromoamine-b) under mild conditions.

Role of the RNA polymerase alpha subunits in CII-dependent activation of the bacteriophage lambda pE promoter: identification of important residues and positioning of the alpha C-terminal domains.

The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream. The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70). The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

Location of the Escherichia coli RNA polymerase α subunit C-terminal domain at an FNR-dependent promoter: analysis using an artificial nuclease

The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase α subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase α subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one α subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second α subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.

Mapping protein-ligand interactions by hydroxyl-radical protein footprinting.

Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high-resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligand.