Micromechanical Measurements on Chemomechanical Protein Aggregates
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0975-DD03-10
Micromechanical Measurements on Chemomechanical Protein Aggregates Stefan Schwan1, Markus Fritzsche1, Andreas Cismak1, Gundula Noll2, Dirk Pr¸fer2, Uwe Spohn1, and Andreas Heilmann1 1 Fraunhofer Institute for Mechanics of Materials Halle, Walter-H¸lse-Strafle 1, Halle (Saale), 06120, Germany 2 Institute for Biochemistry and Biotechnology of Plants, Westf‰lische Wilhelms-Universit‰t M¸nster, Hindenburgplatz 55, M¸nster, 48143, Germany Abstract Chemo-mechanical protein aggregates in plants, e.g. the forisomes of legumes, transform the chemical free energy of their reaction with calcium ions into mechanical energy. Light and scanning electron microscopy analyses demonstrated that the spindle-shaped bodies with lengths between 25 and 40 µm consist of fiber bundle units which change their length and diameter during a reversible switching reaction induced either by calcium, barium or strontium ions, or by changes of the pH value. For the determination of forces generated during the switching reaction, a force measurement in vitro system based on the bending of thin glass fiber was developed. By using this set-up, dynamical forces of forisomes, in the presence of different alkaline earth ions, in the range up to 180 nN were measured. Keywords: forisome; force measurements. 1. Introduction Protein based motors transform chemical free energy into mechanical work [1,2] associated with a conformational change in protein structure. Possible mechanisms are, e.g., protonation of carboxyl or amino groups, or deprotonation of ammonium and carboxylic groups. Besides ATP dependent molecular motors [2], Ca2+-induced protein motors have been investigated, e.g. spasmonemes from vorticella [3-5] and forisomes from legumes [6-8]. Forisomes which are specialized P-protein bodies found within the phloem of legumes transform free reaction energy into mechanical work [6-8]. The spindle-shaped bodies with a length between 25 and 40 µm consist of fiber bundle units which change their length and diameter during the reversible switching reaction. In the presence of free Ca2+, Sr2+, Ba2+ -ions, forisomes contract from longitudinally expanded to a contracted state. The thermodynamic cycle process [8] can be completed by the extraction of these ions with ethylenediaminetetraacetic acid (EDTA) and the forisomes revert to the longitudinally expanded state. In the absence of dissolved oxygen, switching between the longitudinally expanded and the contracted state is reversible and can be repeated several times. In this work, we analyzed the strength and dynamics of forces underlying the conformational change of forisomes in the presence of different ions and showed that Ca2+, Sr2+, and Ba2+ ions induce the generation of approximately the same mechanical force.
2. Materials and methods Forisomes were isolated from Vicia faba as described previously [6]. Ground phloem tissue was stored at ñ 80 ∞C over several weeks. In an M buffer (0.1 M KCl, 10.0 mM TRIS, pH 7.3) with 10 mM EDTA the phloem tissue was defrosted at 20∞C. Thereafter, the sus
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