Native FKBP12 Engineering by Ligand-Directed Tosyl Chemistry: Labeling Properties and Application to Photo-Cross-Linking of Protein Complexes in Vitro and in Living Cells

2012 ◽  
Vol 134 (4) ◽  
pp. 2216-2226 ◽  
Author(s):  
Tomonori Tamura ◽  
Shinya Tsukiji ◽  
Itaru Hamachi
Keyword(s):  
Protein Complexes ◽  
Living Cells ◽  
Cross Linking

Genetically encoded selective cross-linkers and emerging applications

2020 ◽  
Vol 48 (4) ◽  
pp. 1807-1817
Author(s):  
Haiyan Ren
Keyword(s):  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Complexes ◽  
Living Cells ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
New Class ◽  
Selective Reaction ◽  
Biological Studies ◽  
Lower Background

There has been a large amount of interest in the development of genetically encoded cross-linkers that target functional groups naturally present in cells. Recently, a new class of unnatural amino acids that specifically react with target residues were developed and genetically incorporated. The selective reaction shows higher cross-linking efficiency, lower background and predictable cross-linking sites. It has been applied to enhance protein/peptide stability, pinpoint protein–protein interactions, stabilize protein complexes, engineer covalent protein inhibitors, identify phosphatases in living cells, etc. These new covalent linkages provide excellent new tools for protein engineering and biological studies. Their applications in biotherapy will provide considerable opportunities for innovating and improving biomolecular medicines.

scholarly journals The Glycosylphosphatidylinositol-Anchored DFG Family Is Essential for the Insertion of Galactomannan into the β-(1,3)-Glucan–Chitin Core of the Cell Wall of Aspergillus fumigatus

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Laetitia Muszkieta ◽  
Thierry Fontaine ◽  
Rémi Beau ◽  
Isabelle Mouyna ◽  
Marian Samuel Vogt ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Gene Family ◽  
Protein Complexes ◽  
Growth Conditions ◽  
Fungal Cell Wall ◽  
Cross Linking ◽  
Fungal Cell ◽  
Content Type

ABSTRACT The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatus. IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

scholarly journals Identifying hetero-protein complexes in the nuclear envelope

2019 ◽  
Author(s):  
J. Hennen ◽  
K.H. Hur ◽  
J. Kohler ◽  
S.R. Karuka ◽  
I. Angert ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein Complexes ◽  
Living Cells ◽  
Model Systems ◽  
Linc Complex ◽  
Oligomeric State ◽  
Subcellular Compartment ◽  
Dual Color

AbstractThe nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ~40 nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes which are mediated by protein complexes within this subcellular compartment. While fluorescence fluctuation spectroscopy (FFS) is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional FFS methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton (LINC) complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE hetero-protein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies hetero-protein complexes and their stoichiometry in the NE by accounting for spectral crosstalk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the LINC complex, a hetero-protein complex composed of Klarsicht/ANC-1/SYNE homology (KASH) and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the KASH protein nesprin-2 to form a hetero-complex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE hetero-protein complexes.Statement of SignificanceProtein complexes found within the nuclear envelope (NE) play a vital role in regulating cellular functions ranging from gene expression to cellular movement. However, the assembly state of these complexes within their native environment remains poorly understood, which is compounded by a general lack of fluorescence techniques suitable for quantifying the oligomeric state of NE protein complexes. This study aims at addressing this issue by introducing dual-color time-shifted mean-segmented Q analysis as a fluorescence fluctuation method specifically designed to identify the average oligomeric state of hetero-protein complexes within the NE of living cells.

scholarly journals Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking.

1987 ◽  
Vol 105 (2) ◽  
pp. 855-862 ◽  
Author(s):  
R D Balczon ◽  
B R Brinkley
Keyword(s):  
Protein Complexes ◽  
Chinese Hamster ◽  
Protein Band ◽  
Specific Protein ◽  
Microtubule Assembly ◽  
Cross Linking ◽  
Affinity Column ◽  
Metaphase Chromosomes ◽  
In Vitro Assembly

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

Quinolimide-based peptide biosensor for probing p25 in vitro and in living cells

2021 ◽  
pp. 129929
Author(s):  
Francisco Fueyo-González ◽  
Rosario Herranz ◽  
Simona Plesselova ◽  
Maria D. Giron ◽  
Rafael Salto ◽  
...  
Keyword(s):  
Living Cells
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