Hideyuki F.
Arata†
*,
Frederic
Gillot‡
,
Takahiko
Nojima
,
Teruo
Fujii
and
Hiroyuki
Fujita
Institute of Industrial Science (IIS), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan. E-mail: Hideyuk.Arata@curie.fr
First published on 29th July 2008
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.
GFP loses its fluorescence when denatured by temperatures higher than 70 °C,5,6 pH extremes or guanidinium chloride.6 It recovers its fluorescence partially only when renatured.6,7 The thermal stability of isolated and extracted recombinant GFP was evaluated.5 The temperature-dependence of GFP fluorescence from proton wires to proton exit were also studied.8 To elucidate the mechanism of denaturation properties at the molecular level, we need to perform real-time thermal experiments at the millisecond time scale during which these molecular phenomena occur.
Temperature controlling experiments by conventional techniques, such as laser heating, enable a response speed at the subsecond time scale.9,10 However, in those experiments, the experimental setup must be manually adjusted from one run to another. We also need expensive setups which require high skills to operate. This has been the major limitation to conducting enough experiments for statistical analysis. A new experimental procedure and tools for high speed temperature control with simple setups, which enable numbers of experiments at the required place, is necessary for further progress of this research field at the molecular level.
On the other hand, miniaturized devices have realized rapid, highly sensitive and parallel biological or chemical analysis with small amounts of samples thanks to the merits of miniaturization.11–13 An on-chip micro-thermodevice of a few hundred micrometers in size realized enzymatic experiments at high temperatures14 and real-time observation of biomolecular motors15 by rapid temperature alternation with a response time at the 1 second time scale. However, this response time was not fast enough to observe the denaturation of fluorescent proteins. In this report, a micro-thermodevice, which can control the temperature with a response time of 1 millisecond, was manufactured to measure the denaturation dynamics of fluorescent proteins. Hence, those dynamics were successfully measured at the millisecond time scale.
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Fig. 1 The micro-thermodevice for millisecond measurements. (a) A microheater fabricated on a glass plate. The bright part is patterned Ni and the dark part is naked glass. (b) A micro-patterned PDMS sheet. Cylindrical holes are patterned on one side of a PDMS sheet. The bottom right is a expanded picture taken by a scanning electron microscope (SEM). (c) A micro-patterned PDMS sheet was mounted on a microheater fabricated glass plate to form microcontainers. Buffer with proteins in microcontainers were heated up by a collocated microheater. (d) The step response of the microheater shows that this system enables temperature alternation experiments at the millisecond time scale. |
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Fig. 2 FEM simulation of the temperature homogeneity in transient mode. Part (a) represents the device geometry used for the simulation and the temperature distribution at 0.3 ms. Part (b) represents the temperature at the bottom-left corner as well as the top-center of the microcontainers vs. time. The temperatures of the solution in microcontainers are uniform enough at the millisecond time scale. |
The proteins were kept in TE buffer (pH 7.4) at 37 nM. The buffer with proteins was poured on a microheater integrated glass plate. Next, a microfabricated PDMS sheet was mounted on to the buffer drop to form microcontainers as we described in the previous report.12 In this experiment, we used 28 fL PDMS microcontainers. In this volume, one microcontainer contains about 600–700 protein molecules.
The protein shows fluorescence intensity in a microcontainer at room temperature. We induced the energy, which was slightly below that which boils the water, to the microheater. That is to say, in each experiment, the temperatures were raised from room temperature to slightly below 100 °C to observe the quenchings of the fluorescent proteins. Video images were captured by a high speed camera Cascade II: 512 (EMCCD technology) operated at ∼5 ms per frame; this frame rate limits the time resolution in this setup. Fluorescence intensities of the proteins in the microcontainers were analyzed by the image processing software (METAMORF). The shutter of the mercury lamp was opened manually just before switching on the microheater and closed after images were captured. This cycle took a few seconds each time.
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Fig. 3 Time courses of fluorescence intensity of red fluorescent proteins in microcontainers. (a) is for rDsRed and (b) is for rHcRed. Group of curves in graph (a) are from 5 individual microcontainers containing the same concentration of rDsRed. Similarly, the group of curves in graph (b) are from 6 individual microcontainers containing the same concentration of rHcRed. Insets show the same data in a longer time range. The fluorescence intensity was quenched as the microheater was switched on at an elapsed time of 0 second. The quenching completed within 5–10 ms. |
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Fig. 4 Time courses of fluorescence intensity of GFPs in microcontainers. Part (a) is for rAcGFP and part (b) is for rGFPuv. Group of curves in graph (a) are from 5 individual microcontainers containing the same concentration of rAcGFP. Similarly, group of curves in graph (b) are from 6 individual microcontainers containing the same concentration of rGFPuv. The fluorescent intensity of each microcontainer was plotted in line. The average intensity of all the containers is plotted in triangles. The fluorescent intensity quenched when the microheater was switched on at an elapsed time of 0 second. The quenching took longer than in red fluorescent proteins with a bi-exponential curve. |
The quenching curves of both rAcGFP and rGFP were fitted by bi-exponential decay. We used the least square method iteration by commercial software (Origin v7: Origin Lab.) to fit the quenching curve of GFPs as shown in Fig. 5 ((a) is for rAcGFP and (b) is for rGFPuv). Time courses of intensities in rAcGFP and rGFPuv were fitted by
y = A1 exp(−x/τ1) + A2 exp(−x/τ2) +y0.
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Fig. 5 GFPs quenching curves fitted by bi-exponential formula. The raw data of average intensity of all the microcontainers (circle) and bi-exponential fitting curve (continuous line) for rAcGFP (a) and rGFPuv (b). rAcGFP denatured with time constants of 5 ms and 75 ms while rGFPuv denatured with 10 ms and 130 ms. |
The fitted parameters for rAcGFP and rGFP are shown in Table 1. Both in rAcGFP and rGFP, τ1 and τ2 showed a big difference (19 and 14 times difference for rAcGFP and rGFPuv, respectively) which supports the fact that the curves have 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 suggest that GFPs have two states during their denaturation. The bi-exponential decay may be caused by the reverse process of two states renaturation observed by Ueno et al.4 in which a GFP molecule recovered its fluorescence in around 30 s after being denatured by acid. The difference in the time scale between Ueno et al. and our results may be due to the different speed between denaturation and renaturation. It could also be caused by the difference in the extent of denaturation. The parameter difference between rAcGFP and rGFP may be explained by the difference in the position of mutated amino acids.
rAcGFP: | rGFPuv: | ||||
---|---|---|---|---|---|
R 2 = 0.96968 | R 2 = 0.99167 | ||||
Parameter | Value | Error | Parameter | Value | Error |
Y 0 | −1823.60057 | 11.02184 | y 0 | −12938.6833 | 63.43009 |
A 1 | 1230.87943 | 59.32667 | A 1 | 8118.11719 | 228.16326 |
τ 1 | 0.00427 | 4.84811 × 10−4 | τ 1 | 0.00964 | 5.5797 × 10−4 |
A 2 | 591.7941 | 36.07947 | A 2 | 5165.24242 | 139.60186 |
τ 2 | 0.07611 | 0.00824 | τ 2 | 0.12776 | 0.00746 |
This micro-thermodevice can control the temperature and the exposure time to biomaterials down to the millisecond time scale. This system has given the first access to measuring the real-time observation of proteins’ thermal denaturation at the time scale in which those important functions at the molecular level actually happen. The study of thermodynamic properties, such as Gibbs's free energy, at the millisecond time scale will also be possible with this experimental procedure. Furthermore, the device is applicable to other biomaterials such as nucleic acids or proteins. Because the device allows us to perform experiments without any mutations or biochemical treatment to the material itself, it could be used as a general purpose tool for bioresearchers. This microdevice manufactured by microfabrication technologies can be mass produced which facilitates a large number of experiments. The experiments can also be repeated with the same microheater patterned chip by changing PDMS sheets through easy bench top preparation. This compact device allows samples to be analyzed at the point of need which cannot be done with huge equipment. Furthermore, this device can be easily connected to electronic devices for digital sampling or controlling. It also has a potential for on-chip data analysis by integrating an electronic circuit on the same device. Further study with this device may lead to a breakthrough in the field of protein analyses at the molecular level.
Footnotes |
† Research fellow of the Japan Society for the Promotion of Science (JSPS), Tokyo, Japan. |
‡ LIMMS-CNRS/IIS, France/Japan. |
This journal is © The Royal Society of Chemistry 2008 |