Name:Thomas Wendt Position: Pre-doctoral Fellow Qualifications:Diploma, University of Heidelberg (Apr.,1994) Tel. (office): + 49 6221 387241 curriculum vitae

Current Address:

Similar to vertebrate skeletal muscle, the regulatory complex of thin filaments in insect flight muscle consists of tropomyosin (TM) and three troponin components (1). TM and troponin C (TnC) are similar in structure and function to vertebrate. Troponin T (TnT) has a higher molecular weight because of an insertion. Troponin I (TnI) is not detectable in Lethocerus flight muscle. It is replaced by the 80 kD component troponin H (TnH). TnH does not show the same characteristics as vertebrate TnI.

Anti-Lethocerus-TnH antibody recognizes a protein of similar molecular weight in Drosophila flight muscle, which is a fusion protein of tropomyosin and a hydrophobic proline-rich sequence. This region might be extended and form a connection to the thick filaments and could therefore be the mechanical basis for stretch activation in insect flight muscle.

Native troponin complex was isolated and tested for functionality. It was shown by rotary shadowing and paracrystallization on monolayers that the regulatory complex consisted of tropomyosin and the troponin complex. This lollipop-like structure consists of a head region of about 130 A in diameter and a tail region of about 400 A in length. The complex forms paracrystals at the air water interface on a positively charged monolayer. It shows an ordered packing of the globular domain and a more disordered tail. The spacing between rows of packed heads is about 38 nm which corresponds well with the repeating unit of the regulatory complex along insect flight muscle thin filaments.

A three-dimensional model of the troponin complex was reconstructed using the SPIDER software package for single particle analysis.

Paracrystals in solution were grown by a pH precipitation method first described by Bailey for tropomyosin and similar paracrystals were also obtained by adding divalent cations to the troponin-tropomyosin sample. These showed globular domains in a regular pattern along the ribbon-like filaments. The spacing of the repeating unit was determined to be 38 nm which corresponds to the length of tropomyosin-filaments along the actin helix. The paracrystals were analyzed by SDS gel electrophoresis and showed that all subunits of the regulatory complex are included. By varying the concentration of the divalent cations and the protein, it is possible to change the thickness of the ribbons.