scholarly journals The Use of Reductive Methylation of Lysine Residues to Study Protein-Protein Interactions in High Molecular Weight Complexes by Solution NMR

10.5772/38640 ◽  
2012 ◽  
Author(s):  
Youngshim Lee ◽  
Sherwin J. ◽  
Vadim Gaponenko
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Interactions ◽  
Solution Nmr ◽  
Protein Protein Interactions ◽  
Reductive Methylation ◽  
Lysine Residues

scholarly journals TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures inEscherichia coli

1999 ◽  
Vol 181 (18) ◽  
pp. 5563-5571 ◽  
Author(s):  
Heike Schmidt-Eisenlohr ◽  
Natalie Domke ◽  
Christian Baron
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Interactions ◽  
Dna Transfer ◽  
Protein Protein Interactions ◽  
High Molecular Weight Complex ◽  
Plasmid Pkm101 ◽  
Transposon Insertions ◽  
Transfer Apparatus ◽  
Weight Structures

ABSTRACT Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coliallowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,traM, traB, traD, andtraE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essentialtra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

scholarly journals Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

2004 ◽  
Vol 167 (4) ◽  
pp. 699-709 ◽  
Author(s):  
Simona Paladino ◽  
Daniela Sarnataro ◽  
Rudolf Pillich ◽  
Simona Tivodar ◽  
Lucio Nitsch ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Epithelial Cells ◽  
High Molecular Weight ◽  
Protein Interactions ◽  
Mdck Cells ◽  
Protein Oligomerization ◽  
Protein Protein Interactions ◽  
Additional Step ◽  
Apical Sorting

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein–protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

scholarly journals Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Dan Tan ◽  
Qiang Li ◽  
Mei-Jun Zhang ◽  
Chao Liu ◽  
Chengying Ma ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Information ◽  
Affinity Purification ◽  
Protein Protein Interactions ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Lysine Residues ◽  
Spacer Arm ◽  
Cross Linker ◽  
Protein Nucleic Acid

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

Low-molecular-weight spies of protein–protein interactions

2008 ◽  
Vol 11 (4-5) ◽  
pp. 499-505 ◽  
Author(s):  
Jascha Blobel ◽  
Rosa Fayos ◽  
Jesús García ◽  
Oriol Marimon ◽  
Yolanda Pérez ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Protein Interactions ◽  
Low Molecular Weight ◽  
Protein Protein Interactions

scholarly journals An optimized isotopic labelling strategy of isoleucine-γ2methyl groups for solution NMR studies of high molecular weight proteins

2012 ◽  
Vol 48 (10) ◽  
pp. 1434-1436 ◽  
Author(s):  
Isabel Ayala ◽  
Olivier Hamelin ◽  
Carlos Amero ◽  
Ombeline Pessey ◽  
Michael J. Plevin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Solution Nmr ◽  
Isotopic Labelling ◽  
Nmr Studies ◽  
High Molecular Weight Proteins
Sign in / Sign up

Export Citation Format

Share Document