Characterization of Protein Complexes using Targeted Proteomics

2014 ◽  
Vol 14 (3) ◽  
pp. 344-350 ◽  
Author(s):  
Yassel Gomez ◽  
Sebastien Gallien ◽  
Vivian Huerta ◽  
Jan Oostrum ◽  
Bruno Domon ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Targeted Proteomics

scholarly journals Protein complex formation in methionine chain-elongation and leucine biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Li-Qun Chen ◽  
Shweta Chhajed ◽  
Tong Zhang ◽  
Joseph M. Collins ◽  
Qiuying Pang ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Complete Characterization ◽  
Bimolecular Fluorescence Complementation ◽  
Chain Elongation ◽  
Substrate Channeling ◽  
Leucine Biosynthesis ◽  
Protein Complex Formation ◽  
Genes Encoding ◽  
Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

scholarly journals Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

2012 ◽  
Vol 13 (8) ◽  
pp. 10537-10552 ◽  
Author(s):  
Vincent J. B. Ruigrok ◽  
Mark Levisson ◽  
Johan Hekelaar ◽  
Hauke Smidt ◽  
Bauke W. Dijkstra ◽  
...  
Keyword(s):  
Protein Complexes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Alternative Approaches

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein

1990 ◽  
Vol 10 (9) ◽  
pp. 4480-4485
Author(s):  
J Andersen ◽  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Snrnp Core ◽  
Small Nuclear Ribonucleoprotein Particle

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

Characterization of Pt-protein complexes by nHPLC–ESI-LTQ MS/MS using a gel-based bottom-up approach

Talanta ◽  
2012 ◽  
Vol 88 ◽  
pp. 599-608 ◽  
Author(s):  
Estefanía Moreno-Gordaliza ◽  
Benito Cañas ◽  
María A. Palacios ◽  
M. Milagros Gómez-Gómez
Keyword(s):  
Protein Complexes ◽  
Bottom Up

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

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