A new temporal dimension for multisignal sedimentation velocity as a tool to analyze multicomponent interactions

Authors

• SK Chaturvedi
• EA Andrade
• G Patterson
• H Zhao
• P Schuck

Abstract

Multisignal sedimentation velocity run at different wavelength may be a good tool to detect the protein interaction in sedimentation experiment. Reversible enhanced green fluorescent proteins (rsEGFP) are promising tool to track the protein-protein interaction which also facilitate many new exciting application in analytical ultra-centrifugation. rsEGFP2 can be reversibly switched between fluorescent and nonfluorescent state, by irradiating with a specific wavelength of light i.e., 405 nm and 488 nm, respectively. In both states rsEGFP2 has a different absorption maximum (407 and 479 nm). We can use the different absorption wavelength of rsEGFP2 during sedimentation run and exploit the time-dependent shift in absorption to extend multi signal sedimentation velocity boundaries currently used to study multicomponent interactions. As a proof of principle, we observed that absorption sedimentation velocity profiles of rsEGFP2 , with and without illumination with 405 nm light. We performed the sedimentation velocity experiment of rsEGFP2 while irradiated with 405 nm light and read the absorption at 479 and 407 nm. Exponential increase in absorption at 479 nm with rate of 0.573 hour-1 was found as rsEGFP2 attaining the fluorescent state. The results correlate well with the fluorescence detected profiles, which also exhibit the time-dependent signal. This study may helpful to study the heterogeneous multiprotein interactions in the system.

Scientific Focus Area: Biomedical Engineering and Biophysics

This page was last updated on Friday, March 26, 2021