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

  • Katarzyna Tych
  • Gabriel ŽoldákEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Protein elasticity Mini-proteins Engineering Nanomechanics Laser traps 

Notes

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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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Physics Department E22Technical University of MunichGarchingGermany
  2. 2.Center for Interdisciplinary Biosciences, Technology and Innovation ParkP.J. Šafárik UniversityKošiceSlovakia

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