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Stable Substructures in Proteins and How to Find Them Using Single-Molecule Force Spectroscopy

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Protein Supersecondary Structures

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1958))

Abstract

Three-dimensional structures of proteins are a source of fascination for scientists, due to the beauty of their sequence-encoded architectures and their highly diverse range of functions. These functions include acting as powerful catalysts, signal receptors, and versatile molecular motors as well as being building blocks for macroscopic structures, thus defining the shape of multicellular organisms. How protein structure is organized and assembled at the sub-nanometer scale is of great current interest. Specifically, the discovery of stable substructures and supersecondary structures has inspired research into their potential use in rationally engineered proteins with tailor-made properties. Here, we show how the search for stable substructures in large proteins can benefit from recent advances in single-molecule force spectroscopy using highly sensitive dual-beam optical tweezers. Our chapter provides a step-by-step description of the experimental workflow for (1) preparing proteins for mechanical interrogation, (2) interpreting the data, and (3) avoiding the most commonly occurring mistakes.

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Notes

  1. 1.

    During step c, prepare protein coupling premix used in the next step.

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Acknowledgments

The authors have both worked for several years in the single-molecule laboratory of Prof. M. Rief at the Technical University of Munich (TUM), Germany. The various controls and methods that we describe have been developed and optimized by many talented Ph.D. students and Postdocs; in particular, we would like to thank Daniela Bauer, Anja Gieseke, Dr. Ulrike Majdic, Dr. Marco Grison, Dr. Alexander Mehlich, Dr. Markus Jahn, Dr. Johannes Stigler, and Dr. Ziad Ganim for their valuable contributions to the assay development and for many discussions. We thank Dr. Benjamin Pelz for constructing the extraordinary “Duck Trap” optical tweezers setup (no ducks were harmed in the making of this instrument). G.Z. is supported by the CVTI reintegration grant. K.T. is supported by SFB863 from the DFG and a HFSP Cross-Disciplinary Postdoctoral Research Fellowship (LT000150/2015-C).

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Authors and Affiliations

  1. Physics Department E22, Technical University of Munich, Garching, Germany

    Katarzyna Tych

  2. Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University, Košice, Slovakia

    Gabriel Žoldák

Authors
  1. Katarzyna Tych
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  2. Gabriel Žoldák
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Corresponding author

Correspondence to Gabriel Žoldák .

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Editors and Affiliations

  1. Department of Mathematics, Rutgers University, Piscataway, NJ, USA

    Alexander E. Kister

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Tych, K., Žoldák, G. (2019). Stable Substructures in Proteins and How to Find Them Using Single-Molecule Force Spectroscopy. In: Kister, A. (eds) Protein Supersecondary Structures. Methods in Molecular Biology, vol 1958. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9161-7_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9161-7_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9160-0

  • Online ISBN: 978-1-4939-9161-7

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