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At least 55 records · Page 3Linked to original sources
PubMed2026

Library-based, multiplexed strategy for mapping protein interaction networks via crosslinking.

BACKGROUND: Protein-protein interactions are fundamental to cellular function, yet resolving their interaction interfaces and dynamic behaviors in native biological contexts remains challenging, particularly for weak or transient interactions. Crosslinking strategies based on noncanonical amino acids offer an effective means to capture such interactions; however, traditional single-site incorporation provides limited coverage and may overlook critical interaction hotspots. RESULTS: By employing a mutagenesis library, multiple interaction partners and cross-linking sites of a target protein can be simultaneously screened in a single experiment, without prior knowledge of its precise structural or functional features, enabling effective and unbiased analysis of its interaction network. In this study, we constructed an amber codon-scanning mutagenesis library of PSMD10, facilitating independent incorporation of the photocrosslinking ncAA p-azido-phenylalanine at multiple distinct residues. This approach allowed us to systematically interrogate and precisely map potential interaction regions across the protein surface. Coupled with crosslinking mass spectrometry, we identified multiple residues involved in intermolecular interactions, as well as previously unreported interaction partners, including T2FA, TBA1C, and ATRIP. CONCLUSIONS: These findings expand our understanding of PSMD10-associated proteasome interactome, demonstrate a multiplexed strategy for in situ mapping of protein interaction interfaces with broad coverage, and offer a valuable platform for developing therapeutics that target protein-protein interactions.

Protein Interaction Mapping↗
PubMed1979

Bonding erythrocytes to plastic substrates by glow-discharge activation.

A procedure was developed for attaching erythrocytes to various plastics. Plastic disks were exposed to ammonia glow discharge (AGD) and found thereafter to form weak bonds with red cells settling from a dilute saline suspension poured over the disks. When the disks were used to produce a rotating shear flow, complete cell detachment was observed for stresses exceeding 34 dynes/cm2. Far stronger bonding was achieved by treating AGD-bonded cells with glutaraldehyde, and subsequent shearing failed to detach any cells (maximum stress 58 dynes/cm2). The proposed chemical mechanism involves attachment of -NH2 groups on the plastic during AGD, and formation also of carbonyl oxygen later, to provide sites for hydrogen bonding with the cell membrane. Subsequent glutaraldehyde exposure produces crosslinking between disk and cell, as well as fixing the cell in the normal way. Thus, the initial step succeeds in attaching erythrocytes in a deformable condition but rather weakly, while the second step produces rigid cells which are also bonded more strongly.

Ammonia↗
PubMed1978

Modification of erythrocyte physicochemical properties by millimolar concentrations of glutaraldehyde.

The effects of low levels of glutaraldehyde uptake (less than 120 mumol/10(10) cells) on the physicochemical properties of human red blood cells (RBC) were investigated. Salient effects include: by different measures of cell deformability, the extent of glutaraldehyde uptake required to decrease cellular deformability was shown to range from approximately 8 to 30 mumol/10(10) cells; osmotically stressed red cells exhibit complete hemolysis when the level of glutaraldehyde uptake is less than 28 mumol/10(10) cells and no hemolysis when uptake is less than 70 mumol/10(10) cells with the extent of hemolysis decreasing in an approximately linear manner with glutaraldehyde uptake between these limits; glutaraldehyde uptake of up to 58 mumol/10(10) cells does not change the cells' density, mean cell volume or ability to retain potassium.

Aldehydes↗
PubMed1979

Fibrin membrane endowed with biological function. IV. Formation of cross-links between fibrinogen (or fibrin) and ribonuclease by transglutaminase.

Transglutaminase from guinea pig liver catalyzed the formation of cross-links between fibrinogen (or fibrin) and ribonuclease. Using transglutaminase, immoblized ribonuclease was prepared by two separate methods: (1) fibrinogen-ribonuclease conjugates formed by transglutaminase were treated with thrombin to make fibrin membrane bound covalently to the enzyme; (2) fibrin polymer formed from fibrinogen with thrombin was covalently bound to ribonuclease by transglutaminase to make fibrin-ribonuclease conjugates.

Animals↗
PubMed1978

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.

Amino Acids↗
PubMed1978

Cross-linking of nucleosomal histones with monofunctional imidoesters.

Cross-linking experiments with the MONOfunctional imidoester methyl-acetimidate, in the pH range 7.0 - 8.0, on rat liver nucleosomes generate a cross-linking pattern almost identical with the one observed for much longer BIfunctional reagents (e.g. dimethylsuberimidate). Combined cross-linking and trypsin digestion experiments suggest that all or at least the great majority of this cross-linking occurs on trypsin digestible segments (or "tails") of the histones. The formation of oligomers over such extremely short distances and especially the observation of an H3 homodimer suggests a very close proximity of half-nucleosomes.

Animals↗
PubMed1977

[Conjugation of bovine gammaglobulin to L-asparaginase (E. coli) (author's transl)].

Bovine gammaglobulin-L-asparaginase conjugates were prepared with an enzymic activity of about 30 IU/mg. Glutardialdehyde was used as bifunctional reagent for the cross-linking in an optimal concentration of 0.16 mg/10 mg protein. The both components were found in the conjugates in a ratio of 1:1.5. The recovery was about 25% of the initial protein amounts. The antigenic specificity of the globulin component decreased up to 60%, the enzymic activity also up to 60% by the conjugation.

Animals↗
PubMed1979

Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis.

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine carbamoyltransferase, measured by molecular sieving, is 250 000. It is composed of six apparently identical subunits with a molecular weight of 39 000, as determined by cross-linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine carbamoyltransferase is a trimer of 140 000 consisting of three identical subunits with a molecular weight of 40 000. Ornithine carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerable lower efficiency than for putrescine. The kinetic mechanism of putrescine carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead-end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established. The properties of putrescine carbamoyltransferase are compared to the known properties of other carbamoyltransferases. The evolutionary implications of this comparison are discussed.

Carboxyl and Carbamoyl Transferases↗
PubMed1976

[Chemical modification of proteins. 2. Blocking of amino groups and basic amino acids and crosslinking of polypeptide chains in casein and field bean globulin by reaction with dialdehyde starch].

The reaction of dialdehyde starch with casein and field-bean globulin leads to a blocking of the protein amino groups and to a decrease of free lysine, arginine and histidine. Maximum values are reached at high protein concentrations and great molar reagent excess. At best 80-93% of the amino groups or of the available lysine may be blocked in this way. For 1% casein and I and 5% globulin solutions, the pH optimum of the reaction lies at approximately 8; for 5% casein solutions, it is shifted towards the neutral to weakly acidic range. The value for the proportion of unblocked lysine is higher (approximately 5%) when determined by amino-acid analysis after acid total hydrolysis than when measured by means of the colorimetric method according to Carpenter (20%). The difference is designed as reversibly blocked lysine proportion. There is a linear correlation between the proportion of blocked lysine and the relative nutritional value as determined by means of the test organism Tetrahymena pyriformis. Dependently on protein concentration and reagent excess, gel chromatographically detectable cross-linking products of higher molecular weight are formed by the reaction of dialdehyde starch with casein. In 5% protein solutions, such products with molecular weights of less than or equal to 900 000 are the sole detectable components.

Amino Acids↗
PubMed1979

Hydrolysis of p-NN'-phenylenebismaleimide and its adducts with cysteine. Implications for cross-linking of proteins.

To understand the extent of the cross-linking of proteins by the bifunctional reagent p-NN'-phenylenebismaleimide, a quantitative study of competing reactions has been undertaken. The two reactive maleimide rings of the bismaleimide are hydrolysed in mildly alkaline aqueous solutions much more rapidly than is the single maleimide ring of the monofunctional analogue N-ethylmaleimide. The kinetics of hydrolysis are second-order, depending on both imide and hydroxyl ion concentration in the pH range 8-10. The hydrolysis of the first imide ring of the bismaleimide is more rapid than the second, with second-order rate constants of 1600 M-1 . s-1 and 500 M-1 . s-1 respectively, at 25 degrees C. The half-times for hydrolysis of the first and second imide rings at pH 9.0 are therefore only 43s and 140s. Because it renders the maleimide ring unreactive towards cysteine, this rapid hydrolysis can limit the extent of cross-linking of proteins by the bismaleimide.

Chemical Phenomena↗
PubMed1979

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.

4-Nitrophenylphosphatase↗
PubMed1978

Stabilization of the relaxed state of aspartate transcarbamoylase by modification with a bifunctional reagent.

Native aspartate transcarbamoylase from Escherichia coli was modified with the bifunctional reagent tartaryl diazide in the presence of the substrate carbamoyl phosphate and the substrate analog succinate. The product had the same sedimentation coefficient as the native enzyme but showed a marked increase in affinity for the substrate aspartate with a hyperbolic saturation curve. The Michaelis constant for aspartate (7.4 mM) is similar to that estimated for the relaxed state of the enzyme. The high substrate affinity was not produced if modification was conducted in the absence of substrate analogs or with a monofunctional reagent. The modified enzyme was also desensitized towards the allosteric effectors ATP and CTP. It appears to represent a stabilized relaxed state whose conversion to the taut state is presumably prevented by cross-linking.

Aspartate Carbamoyltransferase↗
PubMed1977

Localization of reducing group activity in the structural protein ribbons of the egg capsule of the gastropod mollusc Buccinum undatum L.

Egg capsules of the gastropod molluse Buccinum undatum L. show a staining reaction with silver-methenamine reagent with prior treatment with periodate. The reactivity is specifically localized on the striated structural protein ribbons which form the capsule walls. The staining is in the region of ribbon most resistant to chemical degradation and where the constituent protein monomers are believed to overlap and be cross-linked. Since the proteins contain little cysteine it is likely that the staining originates in aldehyde groups. Staining is abolished by borohydride reduction. It is suggested that the aldehyde groups are a locus of stabilization and cross-linking since previous work has also shown association of an aldehyde secretion with hardening of the capsules.

Aldehydes↗
PubMed1979

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.

Binding Sites↗