Millisecond denaturation dynamics of fluorescent proteins revealed by femtoliter container on micro-thermodevice

Abstract

Real-time observation of biomolecular behavior focusing on high speed temperature response is an essential endeavor for further biological study at the molecular level. This is because most of the important biological functions at the molecular level happen at the sub-second time scale. We used our own on-chip microheaters and microcontainers to observe the denaturation dynamics of fluorescent proteins at the millisecond time scale. The microheater controls the temperature in 1 ms under the microscope. Fluorescent proteins were contained in 28 fL PDMS microcontainers to prevent them from diffusing into the solution. The proteins were denatured by high temperatures and observed by a high speed CCD camera with 5 ms per frame. Hence, denaturation speeds of red fluorescent proteins (rDsRed and rHcRed) were measured to be 5–10 ms. Green fluorescent proteins (rAcGFP and rGFPuv) denatured with bi-exponential decay. rAcGFP denatured with time constants of 5 ms and 75 ms while rGFPuv denatured with 10 ms and 130 ms. This may be the reverse process of a two step renaturation of GFP observed in a previous report. This micro-thermodevice is applicable to other biomaterials such as nucleic acids or other proteins. It does not require any chemical treatment nor mutation to the biomaterial itself. Therefore, the methodology using this general purpose device gives access to biomolecular studies in short time scales and acts as a powerful tool in molecular biology.