Probing conformational changes of prestin with thiol-reactive optical switches.
Thiol-reactive optical switch probes were used to examine conformational changes of prestin-based membrane motor. Because this motor is based on mechanoelectric coupling similar to piezoelectricity, the motile activity can be monitored by charge movements across the plasma membrane, which appears as nonlinear capacitance. When the plasma membrane is conjugated with the probes, optically induced spiro-merocyanine transition positively shifted nonlinear capacitance of outer hair cells and prestin-transfected cells by approximately 10 mV. These shifts were reversible and were eliminated by pretreatment with iodoacetamide. However, they were little affected by pretreatment with biotin maleimide, which cannot reach the cytoplasmic surface. Our results showed that merocyanine states, with a larger dipole moment, interact with the motor's extended conformation stronger than with the compact conformation by 1.6 x 10(-21) J/molecule. The interaction sites are near the cytoplasmic side of the motor protein.
Biophysics Section, Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland, USA.
Jie
J
Fang
Tomoyo
T
Sakata
Gerard
G
Marriott
Kuni H
KH
Iwasa
D000818:Animals; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D025802:Electric Capacitance; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D008301:Maleimides / Q000737:chemistry / Q000494:pharmacology; D055095:Optics and Photonics; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D027321:Anion Transport Proteins; D008301:Maleimides; D013438:Sulfhydryl Compounds; C043592:maleimide; D001710:Biotin; D003545:Cysteine; D007460:Iodoacetamide