1. Structural Biology and Molecular Biophysics

Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

  1. Anusmita Sahoo
  2. Shruti Khare
  3. Sivasankar Devanarayanan
  4. Pankaj Jain
  5. Raghavan Varadarajan  Is a corresponding author
  1. Indian Institute of Science, India
https://doi.org/10.7554/eLife.09532
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  1. Anusmita Sahoo
  2. Shruti Khare
  3. Sivasankar Devanarayanan
  4. Pankaj Jain
  5. Raghavan Varadarajan
(2015)
Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis
eLife 4:e09532.
https://doi.org/10.7554/eLife.09532
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Abstract

Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in-vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.

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Author details

  1. Anusmita Sahoo

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
    Competing interests
    Anusmita Sahoo, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.

  2. Shruti Khare

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
    Competing interests
    Shruti Khare, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.

  3. Sivasankar Devanarayanan

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
    Competing interests
    No competing interests declared.

  4. Pankaj Jain

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
    Competing interests
    Pankaj Jain, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.

  5. Raghavan Varadarajan

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
    For correspondence
    varadar@mbu.iisc.ernet.in
    Competing interests
    Raghavan Varadarajan, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.

Copyright

© 2015, Sahoo et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Anusmita Sahoo
  2. Shruti Khare
  3. Sivasankar Devanarayanan
  4. Pankaj Jain
  5. Raghavan Varadarajan
(2015)
Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis
eLife 4:e09532.
https://doi.org/10.7554/eLife.09532

Share this article

https://doi.org/10.7554/eLife.09532

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