Measuring biological materials mechanics with atomic force microscopy ‐ Mechanical unfolding of biopolymers

• Published in: Microscopy research and technique Vol. 85; no. 8; pp. 3025 - 3036
• Main Authors: Gil‐Redondo, Juan Carlos, Weber, Andreas, Toca‐Herrera, José L.
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.08.2022; Wiley Subscription Services, Inc
• Subjects: Atomic force microscopy; Biological materials; Biopolymers; Connectin; energy landscape; Force measurement; I27; Macromolecules; Mechanical properties; mechanical unfolding; Microscopy; Muscles; Polymers; Polynucleotides; Polypeptides; Polysaccharides; Proteins; Saccharides; Spectroscopy; Spring constant; Stability; thermal softening; titin
• ISSN: 1059-910X; 1097-0029
• DOI: 10.1002/jemt.24136

Biopolymers, such as polynucleotides, polypeptides and polysaccharides, are macromolecules that direct most of the functions in living beings. Studying the mechanical unfolding of biopolymers provides important information about their molecular elasticity and mechanical stability, as well as their energy landscape, which is especially important in proteins, since their three‐dimensional structure is essential for their correct activity. In this primer, we present how to study the mechanical properties of proteins with atomic force microscopy and how to obtain information about their stability and energetic landscape. In particular, we discuss the preparation of polyprotein constructs suitable for AFM single molecule force spectroscopy (SMFS), describe the parameters used in our force‐extension SMFS experiments and the models and equations employed in the analysis of the data. As a practical example, we show the effect of the temperature on the unfolding force, the distance to the transition state, the unfolding rate at zero force, the height of the transition state barrier, and the spring constant of the protein for a construct containing nine repeats of the I27 domain from the muscle protein titin. Highlights 1. Atomic force microscopy (AFM) can be used to study the mechanical unfolding of polymers. 2. AFM provides a direct measurement of unfolding (unbinding) forces. 3. Force measurements for different rates provide information about the distance to the transition state and the unfolding rate at zero force. Mechanical unfolding of (poly) proteins can be studied with atomic force microscopy. Above: sequential unfolding of the individual proteins until the (poly)protein is completely stretched and detached from the tip. Below: energy landscape that can be investigated with such experiments.