Structural analysis of the motor protein prestin

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The aim of this project was to clarify the motor protein prestin, whose conformational change has been indicated to induce an elongation and contraction of outer hair cells in the inner ear. To this end, first, an attempt was made to observe the plasma membrane of prestin-transfected Chinese hamster ovary (CHO) cells and that of untransfected CHO cells by atomic force microscopy (AFM). As a result, particle-like structures probably indicating membrane proteins were recognized in the plasma membranes of both cells, although there were no distinctive differences between the two. Analysis of the shape and size of the observed structures showed that there were more particle-like structures with a diameter of 8-12 nm in the plasma membrane of the prestin-transfected CHO cells than in that of the untransfected CHO cells, suggesting that these particle-like structures are possibly prestin molecules.The second endeavor was to more specifically detect prestin molecules in the plasma membrane of the prestin-transfected CHO cells to clarify the structure of prestin in more detail. For this purpose, an experimental approach combining AFM with anti-prestin antibody conjugated with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. As Qdots are hard material with a diameter of 8 nm, they can be easily seen by AFM. In addition, as prestin may exist near Qdots, detection and observation of prestin molecules can be performed. As a result of imaging by AFM of the prestin-transfected CHO cells labeled with Qdots, squarish ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, indicating that prestin forms a tetramer in the plasma membranes of the prestin-transfected CHO cells.