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

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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.

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Acknowledgements

We gratefully acknowledge financial support from the National Institute of General Medical Sciences (NIGMS Protein Structure Initiative grant U54GM094597 (GTM), biotechnology resource Grant P41GM103390 and Grant R01GM033225 (JHP)) and from the HHMI (DAA). Molecular graphics and structural analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). Manuscript content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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    Authors and Affiliations

    1. Complex Carbohydrate Research Center, University of Georgia, Athens, USA

      Kari Pederson, Gordon R. Chalmers, Qi Gao & James H. Prestegard

    2. Department of Computer Science, University of Georgia, Athens, USA

      Gordon R. Chalmers

    3. Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, USA

      Daniel Elnatan & David A. Agard

    4. Department of Chemistry and Biochemistry, Miami University, Oxford, USA

      Theresa A. Ramelot & Michael A. Kennedy

    5. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, The State University of New Jersey, Piscataway, USA

      Li-Chung Ma & Gaetano T. Montelione

    6. Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, USA

      Li-Chung Ma & Gaetano T. Montelione

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    1. Kari Pederson
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    Correspondence to James H. Prestegard.

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    Kari Pederson and Gordon R. Chalmers have contributed equally to the work.

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    Pederson, K., Chalmers, G.R., Gao, Q. et al. NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine. J Biomol NMR 68, 225–236 (2017). https://doi.org/10.1007/s10858-017-0123-8

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