Protein Interactions in the Escherichia coli Cytosol: An Impediment to In-Cell NMR Spectroscopy.
Chembiochem. 2011 Mar 29;
Authors: Crowley PB, Chow E, Papkovskaia T
Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15) N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.
PMID: 21448871 [PubMed - as supplied by publisher]
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Biochemistry. 2011 Sep 26;
Authors: Wang Q, Zhuravleva A, Gierasch LM
Abstract
Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods are needed that preserve these interactions...
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09-30-2011 06:00 AM
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell NMR spectroscopy.
Biochemistry. 2011 Sep 26;
Authors: Wang Q, Zhuravleva A, Gierasch LM
Abstract
Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods are needed that preserve these...
nmrlearner
Journal club
0
09-30-2011 05:59 AM
[NMR paper] Solution-state NMR investigation of DNA binding interactions in Escherichia coli form
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DNA Repair (Amst). 2005 Mar 2;4(3):327-39
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11-24-2010 11:14 PM
[NMR paper] Optimization of an Escherichia coli system for cell-free synthesis of selectively N-l
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[NMR paper] 19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate redu
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Biochemistry. 1994 May 10;33(18):5502-9
Authors: Hoeltzli SD, Frieden C
Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure. In order to examine the regions of the protein surrounding these tryptophan...
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08-22-2010 03:33 AM
[NMR paper] 19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate redu
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Biochemistry. 1994 May 10;33(18):5502-9
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http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide. NMR studies using selectively 15N-labelled protein.
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Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)
Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)
David S Burz, Kaushik Dutta, David Cowburn & Alexander Shekhtman
We describe a high-throughput in-cell nuclear magnetic resonance (NMR)-based method for mapping the structural changes that accompany protein-protein interactions (STINT-NMR). The method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring the protein interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define...
Protein Interactions in the Escherichia coli Cytosol: An Impediment to In-Cell NMR Spectroscopy.
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