Abstract
Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916-19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bullet-shaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 x 77 x 115 A, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.
Highlights
The mammalian ear has specialized function in collecting sound signals and transmitting them to the nerve
Expression and Purification of the Prestin Protein—Natural expression of prestin is limited to the outer hair cells of the inner ear and is too scarce for purification
As C-terminally tagged prestin expressed at higher efficiency, we have chosen this construct for further study
Summary
3D, three-dimensional; AFM, atomic force microscopy; DDM, n-dodecyl -D-maltoside; FSC, Fourier shell correlation; GNG, growing neural gas network; MRA, multireference alignment; NN, neural network; OHCs, outer hair cells; RS, Stokes radius; SA, simulated annealing; SEC, size exclusion chromatography; TBS, Tris-buffered saline. Prestin locates on the lateral membrane of the OHC in high densities [19, 20] and in much lower densities on the rest of the membrane, including the basal membrane [21] It has been observed as highly accumulated membrane integral proteins (2,500 particles/m2) about 11 nm in diameter by using the freeze-fracture technique [22] and atomic force microscopy (AFM) [23]. Structural information of prestin is necessary for understanding the generation of the force and its regulation mechanisms To address this issue, we purified recombinant prestin protein from baculovirus-infected Sf9 cells, observed the negatively stained molecules using EM, and reconstructed the 3D structure by single particle analysis [25,26,27]
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