Document Type
Conference Proceeding
Publication Date
4-25-2006
Abstract
The centroid of a fluorophore can be determined within 1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin averaged 128 while the LCD was in the leading state and 57 in the trailing state. The angular difference of 71 represents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When changes, the probe rotates azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of non tilting lever arms that were reported.
Keywords
actin, calmodulin derivative, animal cell, animal tissue, brain tissue, chicken, defocused orientation and position imaging, animals, biophysics, calmodulin, chickens, fluorescent dyes, models, molecular, molecular motor proteins, myosin type v, quantum dots, rhodamines, animalia, accuracy, article, three dimensional imaging, rhodamine, protein structure, protein domain, priority journal, orientation, nonhuman, myosin v, molecular imaging, fluorescence, fluorescence imaging with one nanometer accuracy
Publication Title
Proceedings of the National Academy of Sciences of the United States of America
Rights
© 2006 by The National Academy of Sciences of the USA. Permission granted, contingent on providing a link somewhere in the record to the PNAS Online version as well, and that the content is free and openly available without charge for users: http://www.pnas.org/content/103/17/6495
Recommended Citation
Toprak, Erdal, Joerg Enderlein, Sheyum Syed, Sean A. McKinney, Rolfe G. Petschek, Taekjip Ha, Yale E. Goldman, and Paul R. Selvin. "Defocused orientation and position imaging (DOPI) of myosin V." Proceedings of the National Academy of Sciences 103, no. 17 (2006): 6495-6499.