scholarly journals The interaction between subunits in the tubulin dimer

1985 ◽  
Vol 230 (2) ◽  
pp. 551-556 ◽  
Author(s):  
L Serrano ◽  
J Avila
Keyword(s):  
Formic Acid ◽  
Limited Proteolysis ◽  
Trypsin Digestion ◽  
Cross Linking ◽  
Β Subunit ◽  
Α Subunit ◽  
Tubulin Dimer ◽  
Cross Linker ◽  
Chemical Cross Linking ◽  
Β Tubulin

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between α- and β-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the α-subunit into two fragments, whereas chymotrypsin cleaved the β-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the α-tubulin N-terminal fragment with β-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.

scholarly journals Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

2020 ◽  
Author(s):  
Jan Fiala ◽  
Zdeněk Kukačka ◽  
Petr Novák
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Water Soluble ◽  
Cross Linking ◽  
Side Chains ◽  
Model Protein ◽  
Cross Linker ◽  
Progressive Technology ◽  
The Impact ◽  
Chemical Cross Linking

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

Detection of a Myc-associated protein by chemical cross-linking

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

Thyroid hormone regulates tubulin expression in mammalian liver. Effects of deleting thyroid hormone receptor-α or -β

2005 ◽  
Vol 289 (1) ◽  
pp. E87-E94 ◽  
Author(s):  
Carmen G. Vallejo ◽  
Ana M. Seguido ◽  
Pilar S. Testillano ◽  
María-Carmen Risueño
Keyword(s):  
Thyroid Hormone ◽  
Thyroid Hormone Receptor ◽  
Positive Control ◽  
Β Subunit ◽  
Α Subunit ◽  
Mammalian Liver ◽  
Microtubular Network ◽  
Tubulin Protein ◽  
Β Tubulin

Microtubules are made from polymers of α/β dimers. We have observed in rat liver that, on the first day after birth, α-subunit is relatively high and β-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially β-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TRα or TRβ compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, β-tubulin protein expression in the liver is primarily under TRβ positive control. In euthyroid mice lacking TRβ, β-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TRα might also upregulate β-tubulin expression. Accordingly, TH administration to hypothyroid TRβ-deprived mice reduced their high β-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T3 treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in β-tubulin in mice lacking TRβ. In conclusion, our results indicate that β-tubulin is critically controlled by TRβ in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.

scholarly journals Gelatin-Based Hydrogels through Homobifunctional Triazolinediones Targeting Tyrosine Residues

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 589 ◽  
Author(s):  
Roberto Guizzardi ◽  
Luca Vaghi ◽  
Marcello Marelli ◽  
Antonino Natalello ◽  
Ivan Andreosso ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Drug Delivery Systems ◽  
Chemical Nature ◽  
Mechanical Stability ◽  
Cross Linking ◽  
Swelling Properties ◽  
Tyrosine Residues ◽  
Cross Linker ◽  
The Cross ◽  
Chemical Cross Linking

Gelatin is a biopolymer with interesting properties that can be useful for biomaterial design for different applications such as drug delivery systems, or 3D scaffolds for tissue engineering. However, gelatin suffers from poor mechanical stability at physiological temperature, hence methods for improving its properties are highly desirable. In the present work, a new chemical cross-linking strategy based on triazolinedione ene-type chemistry towards stable hydrogel is proposed. Two different homobifunctional 1,2,4-triazoline-3,5(4H)-diones, namely 4,4′-hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione) 1 and 4,4′-[methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione) 2 were used as cross-linkers in different ratio to tyrosine residues in gelatin. The reaction was proved effective in all experimented conditions and hydrogels featured with different thermal stability were obtained. In general, the higher the cross-linker/tyrosine ratio, the more thermostable the hydrogel. The swelling properties are strictly dependent upon the chemical nature of the cross-linker.

scholarly journals Detection of a Myc-associated protein by chemical cross-linking.

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868 ◽  
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

scholarly journals Tubulin Subunits Exist in an Activated Conformational State Generated and Maintained by Protein Cofactors

1997 ◽  
Vol 138 (4) ◽  
pp. 821-832 ◽  
Author(s):  
Guoling Tian ◽  
Sally A. Lewis ◽  
Becket Feierbach ◽  
Timothy Stearns ◽  
Heidi Rommelaere ◽  
...  
Keyword(s):  
Complex Formation ◽  
Genetic Analysis ◽  
Conformational State ◽  
The Other ◽  
Β Subunit ◽  
Α Subunit ◽  
Folding Intermediates ◽  
Integrated Study ◽  
Β Tubulin

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

scholarly journals Human leucocyte glycosylasparaginase is an α/β-heterodimer of 19 kDa α-subunit and 17 and 18 kDa β-subunit

1994 ◽  
Vol 300 (2) ◽  
pp. 541-544 ◽  
Author(s):  
O K Tollersrud ◽  
T Heiskanen ◽  
L Peltonen
Keyword(s):  
Molecular Mass ◽  
Reversed Phase ◽  
Alpha Subunit ◽  
Cross Linking ◽  
Terminal Sequence ◽  
Α Subunit ◽  
Sds Page ◽  
Specific Antisera ◽  
Purification Scheme ◽  
Chemical Cross Linking

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.

scholarly journals Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

2018 ◽  
Vol 115 (44) ◽  
pp. 11162-11167 ◽  
Author(s):  
Bing Yang ◽  
Haifan Wu ◽  
Paul D. Schnier ◽  
Yansheng Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Current Method ◽  
Thermal Fluctuations ◽  
Limited Range ◽  
Live Cells ◽  
Cross Linking ◽  
Cross Links ◽  
Cross Linker ◽  
Sulfonyl Fluoride ◽  
Chemical Cross Linking

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a “plant-and-cast” cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues “planting” the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then “cast” across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme–substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

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