Journal of Biomolecular NMR

, Volume 68, Issue 3, pp 225–236 | Cite as

NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine

  • Kari Pederson
  • Gordon R. Chalmers
  • Qi Gao
  • Daniel Elnatan
  • Theresa A. Ramelot
  • Li-Chung Ma
  • Gaetano T. Montelione
  • Michael A. Kennedy
  • David A. Agard
  • James H. Prestegard
Article

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.

Keywords

Resonance assignments Assignment program Sparse labeling Perdeuteration Heat-shock protein Hsp90 HtpG Protein structure 1H–13C Ala methyl RDCs Methyl-TROSY 

Supplementary material

10858_2017_123_MOESM1_ESM.pdf (191 kb)
Supplementary material 1 (PDF 191 KB)

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Kari Pederson
    • 1
  • Gordon R. Chalmers
    • 1
    • 2
  • Qi Gao
    • 1
  • Daniel Elnatan
    • 3
  • Theresa A. Ramelot
    • 4
  • Li-Chung Ma
    • 5
    • 6
  • Gaetano T. Montelione
    • 5
    • 6
  • Michael A. Kennedy
    • 4
  • David A. Agard
    • 3
  • James H. Prestegard
    • 1
  1. 1.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  2. 2.Department of Computer ScienceUniversity of GeorgiaAthensUSA
  3. 3.Department of Biochemistry and Biophysics, Howard Hughes Medical InstituteUniversity of CaliforniaSan FranciscoUSA
  4. 4.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA
  5. 5.Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and MedicineThe State University of New JerseyPiscatawayUSA
  6. 6.Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, RutgersThe State University of New JerseyPiscatawayUSA

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