Related ArticlesNMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with (13)C-methyl alanine.
J Biomol NMR. 2017 Jun 26;:
Authors: Pederson K, Chalmers GR, Gao Q, Elnatan D, Ramelot TA, Ma LC, Montelione GT, Kennedy MA, Agard DA, Prestegard JH
Abstract
A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145*kDa dimer. It uses (13)C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from (1)H-(13)C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.
PMID: 28653216 [PubMed - as supplied by publisher]
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine
Abstract
A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145Â*kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1Hâ??13C residual dipolar...
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06-27-2017 03:27 AM
[NMR paper] Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
J Biomol NMR. 2016 Jun 2;
Authors: Monneau YR, Ishida Y, Rossi P, Saio T, Tzeng SR, Inouye M, Kalodimos CG
Abstract
A simple and cost effective method to independently and stereo-specifically incorporate -methyls in Leu and Val in proteins is presented. Recombinant proteins...
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06-04-2016 11:08 AM
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications
Abstract
A simple and cost effective method to independently and stereo-specifically incorporate -methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The...
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06-03-2016 04:52 PM
[NMR paper] CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
Related Articles CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
J Biomol NMR. 2015 Nov 13;
Authors: Kerfah R, Hamelin O, Boisbouvier J, Marion D
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic...
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11-15-2015 07:55 PM
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13Câ??13C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu)...
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11-14-2015 03:37 PM
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Abstract
Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L...
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11-28-2014 11:37 AM
[NMR paper] Sparse labeling of proteins: Structural characterization from long range constraints
Sparse labeling of proteins: Structural characterization from long range constraints
Publication date: April 2014
Source:Journal of Magnetic Resonance, Volume 241</br>
Author(s): James H. Prestegard , David A. Agard , Kelley W. Moremen , Laura A. Lavery , Laura C. Morris , Kari Pederson</br>
Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In...
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03-22-2014 01:28 AM
[NMR paper] High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Applicati
High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.
Related Articles High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.
Biochemistry. 2001 May 29;40(21):6275-83
Authors: Peterson FC, Gordon NC, Gettins PG
We describe here the high-level expression of bovine trypsinogen in E. coli, its...
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with (13)C-methyl alanine.
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