Article | Published:

Electric-field-stimulated protein mechanics

Nature volume 540, pages 400405 (15 December 2016) | Download Citation

Abstract

The internal mechanics of proteins—the coordinated motions of amino acids and the pattern of forces constraining these motions—connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

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Acknowledgements

R.R. dedicates this paper to Alfred G. Gilman, whose contributions were profound and irreplaceable. We thank the staff at BioCARS, Stanford Synchrotron Radiation Lightsource (SSRL) and the UT Southwestern Medical Center Structural Biology Laboratory for technical support, and D. Borek, C. A. Brautigam, S. Leibler, A. Libchaber, K. Moffat, Z. Otwinowski and members of the Ranganathan laboratory for discussions. R.R. acknowledges support from National Institutes of Health (NIH) grant R01GM123456, the Robert A. Welch Foundation (I-1366), the Lyda Hill Endowment for Systems Biology, and the Green Center for Systems Biology. BioCARS is supported by NIH grant R24GM111072 and through a collaboration with P. Anfinrud (NIH/ National Institute of Diabetes and Digestive and Kidney Diseases). The SSRL is supported by the US Department of Energy (Contract No. DE-AC02-76SF00515) and by the NIH (P41GM103393).

Author information

Author notes

    • Doeke R. Hekstra

    Present address: Department of Molecular and Cellular Biology and School of Engineering and Applied Sciences, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, USA.

Affiliations

  1. Green Center for Systems Biology, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA

    • Doeke R. Hekstra
    • , K. Ian White
    • , Michael A. Socolich
    •  & Rama Ranganathan
  2. Center for Advanced Radiation Sources, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA

    • Robert W. Henning
    •  & Vukica Šrajer
  3. Departments of Biophysics and Pharmacology, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA

    • Rama Ranganathan

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Contributions

D.R.H. and R.R. conceived the experimental approach. All authors contributed to the experimental design, D.R.H. and K.I.W. built the EF-X apparatus, and D.R.H., K.I.W., M.A.S. and R.R. performed experiments. D.R.H., V.S. and R.R. developed analysis methods and analysed the data. D.R.H. and R.R. wrote the manuscript with input from the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Rama Ranganathan.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary Discussion, Supplementary Tables 1-7, full legends for PyMol Session files 1-4 and Supplementary References – see contents page for full details.

Zip files

  1. 1.

    Supplementary Data

    This file contains PyMol Session S1, internal electron density difference map shown on the OFF structure – see Supplementary Information document for full description.

  2. 2.

    Supplementary Data

    This file contains PyMol Session S2, superimposed up, down, and OFF models and 2Fo-Fc electron density – see Supplementary Information document for full description.

  3. 3.

    Supplementary Data

    This file contains PyMol Session S3, superimposed up, down, and OFF models and 2Fo-Fc electron density for a composite omit map calculated with iterative refinement – see Supplementary Information document for full description.

  4. 4.

    Supplementary Data

    This file contains PyMol Session S4, 2Fo-Fc electron density obtained for refinement against extrapolated structure factors in the reduced symmetry (P1) space group – see Supplementary Information document for full description.

Videos

  1. 1.

    Destructive breakdown after dielectric seal failure

    An experiment in which misalignment of electrodes led to dissociation of the protein crystal (lysozyme) from the bottom electrode, providing a conductive path around the crystal and leading to destructive arcing (dielectric breakdown). The video was recorded from a monitor with a piece of transparent adhesive tape with red dot indicating beam center, used in sample alignment.

  2. 2.

    Establishing liquid junction between top counter electrode and the crystal

    The approach of the top electrode by manual control of a translation stage. The electrode is brought in sufficiently close to establish a liquid junction between the top electrode and the crystal.

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DOI

https://doi.org/10.1038/nature20571

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