NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine.
J Biomol NMR
; 68(3): 225-236, 2017 Jul.
Article
en En
| MEDLINE
| ID: mdl-28653216
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Coloración y Etiquetado
/
Isótopos de Carbono
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Proteínas HSP90 de Choque Térmico
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Resonancia Magnética Nuclear Biomolecular
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Proteínas de Escherichia coli
/
Alanina
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Biomol NMR
Asunto de la revista:
BIOLOGIA MOLECULAR
/
DIAGNOSTICO POR IMAGEM
/
MEDICINA NUCLEAR
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos