15N-(1)H NMR spectroscopy has been used to probe the dynamic properties of uniformly (15)N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex ( approximately 2.3 MDa), [(1)H-(15)N] heteronuclear single-quantum correlation spectra contain approximately 100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7/L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T(1)) and T(1 rho) (15)N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7/L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7/L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7/L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis.
[NMR paper] (1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamba
(1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles.
Related Articles (1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles.
Biophys J. 2004 Aug;87(2):1205-14
Authors: Metcalfe EE, Zamoon J, Thomas DD, Veglia G
We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid...
nmrlearner
Journal club
0
11-24-2010 10:01 PM
[NMR paper] Rapid screening of E. coli extracts by heteronuclear NMR.
Rapid screening of E. coli extracts by heteronuclear NMR.
Related Articles Rapid screening of E. coli extracts by heteronuclear NMR.
Curr Protoc Protein Sci. 2003 May;Chapter 7:Unit 7.11
Authors: Gronenborn AM
Assessing whether a protein or protein complex is amenable to structural analysis is an important component in the structural genomics effort. In particular, if complete sets of structures for entire genomes are to be obtained within a reasonable time frame, high throughput methodologies for all steps along the way have to be developed....
nmrlearner
Journal club
0
11-24-2010 09:01 PM
[NMR paper] Functionally significant mobile regions of Escherichia coli SecA ATPase identified by
Functionally significant mobile regions of Escherichia coli SecA ATPase identified by NMR.
Related Articles Functionally significant mobile regions of Escherichia coli SecA ATPase identified by NMR.
J Biol Chem. 2002 Dec 27;277(52):50985-90
Authors: Chou YT, Swain JF, Gierasch LM
SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the...
nmrlearner
Journal club
0
11-24-2010 08:58 PM
[NMR paper] NMR structure of ribonuclease HI from Escherichia coli.
NMR structure of ribonuclease HI from Escherichia coli.
Related Articles NMR structure of ribonuclease HI from Escherichia coli.
Biol Pharm Bull. 2000 Oct;23(10):1147-52
Authors: Fujiwara M, Kato T, Yamazaki T, Yamasaki K, Nagayam K
The solution structure of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), a protein of 155 residues, was determined. Three-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain 1,424 distance constraints between individually assigned polypeptide chain hydrogen atoms....
nmrlearner
Journal club
0
11-19-2010 08:29 PM
[NMR paper] Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that
Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution.
J Mol Biol. 1997 Feb 14;266(1):15-22
Authors: Moreau M, de Cock E, Fortier PL, Garcia C, Albaret C, Blanquet S, Lallemand JY, Dardel F
Initiation...
nmrlearner
Journal club
0
08-22-2010 03:31 PM
[NMR paper] Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that
Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution.
J Mol Biol. 1997 Feb 14;266(1):15-22
Authors: Moreau M, de Cock E, Fortier PL, Garcia C, Albaret C, Blanquet S, Lallemand JY, Dardel F
Initiation...
nmrlearner
Journal club
0
08-22-2010 03:03 PM
[NMR paper] The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli pho
The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.
FEBS Lett. 1993 Jan 2;315(1):11-5
Authors: van Nuland NA, Kroon GJ, Dijkstra K, Wolters GK, Scheek RM, Robillard GT
The region of the surface of...
nmrlearner
Journal club
0
08-21-2010 11:53 PM
[NMR paper] NMR studies of the activation of the Escherichia coli trp repressor.
NMR studies of the activation of the Escherichia coli trp repressor.
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 NMR studies of the activation of the Escherichia coli trp repressor.
Eur J Biochem. 1991 Nov 1;201(3):569-79
Authors: Hyde EI, Ramesh V, Frederick R, Roberts GC
The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues...