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NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine
- Source :
- Journal of Biomolecular NMR. 68:225-236
- Publication Year :
- 2017
- Publisher :
- Springer Science and Business Media LLC, 2017.
-
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 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 1H–13C 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.
- Subjects :
- 0301 basic medicine
Alanine
biology
Chemistry
Chemical shift
Dimer
Resolution (electron density)
Crystal structure
010402 general chemistry
Resonance (chemistry)
01 natural sciences
Biochemistry
0104 chemical sciences
03 medical and health sciences
Crystallography
chemistry.chemical_compound
030104 developmental biology
Protein structure
biology.protein
Protein G
Spectroscopy
Subjects
Details
- ISSN :
- 15735001 and 09252738
- Volume :
- 68
- Database :
- OpenAIRE
- Journal :
- Journal of Biomolecular NMR
- Accession number :
- edsair.doi...........c268c4006b0576af55a17329b6e91afd