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A microchannel solution mixer for studying microsecond protein folding reactions
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View: Figures


Image of FIG. 1.
FIG. 1.

Schematic of mixer construction. The -thick PEEK or polyimide mixer having dimensions is sandwiched between two -thick fused silica windows. One of the windows has three holes and channels etched on its surface for fluid delivery and removal. Teflon™ gaskets ( or thick) form a seal between the windows and the stainless-steel holder. Connections to the holder are made via standard 10-32 HPLC fittings. To minimize distortion of the PEEK or polyimide, the length of the input channels of the mixer are typically shorter (typically several mm) than what is shown in the figure. In this case, channels for fluid delivery are made on the window. The direction of flow in the PEEK mixer is indicated by the arrows.

Image of FIG. 2.
FIG. 2.

Experimental arrangement showing the connections between the pumps, protein, NATA sample loops, mixer, and the two different detection modes. For protein refolding experiments, both pumps contain buffer. For NATA quenching experiments, pump B contains the NBS solution instead of buffer. The steady-state detection (top box) utilizes a CCD camera and in-line detection scheme; the time-resolved detection (lower box) is done in epifluorescence mode. The tubing with connector P switches between protein, NATA, and buffer, whereas the tubing with label B always contains the dilution solution. Abbreviations: , , mirror, , tube, , .

Image of FIG. 3.
FIG. 3.

(Color) Mixing images of urea and water. The images are false color representations of light transmission through the channel as viewed under a microscope at magnification. Urea flows into the central horizontal channel from the lower left and buffer from the upper left. The sequence of images are acquired at increasing flow rates showing the transition to turbulent flow. The arrow indicates the distance (time) of complete mixing. Only of the full channel is shown. The Reynolds number at is 2000.

Image of FIG. 4.
FIG. 4.

Kinetics of quenching of NATA fluorescence by varying concentrations of NBS. The final concentrations of NBS are (a) , (b) , and (c) . Data collected in the absence of urea are represented by filled circles and data acquired by diluting from urea are shown by open circles. The solid and dashed lines are local fits to the data in the absence and presence of urea, respectively, using a single exponential decay function with time constants of (a) (filled circles), (open circles), (b) (filled circles), (open circles), and (c) (filled circles), (open circles). The data were acquired using TCSPC fluorescence detection and a flow rate of . The zero time of the reaction corresponds to the apex of the channel. The dotted vertical line at represents the time for complete mixing and typically the first usable data point. The NATA concentration was at pH 7.0 and . The fluorescence was collected using a monochromator set at with a bandwidth. The excitation wavelength was . Each trace represents the average of of data collection. The signal-to-noise ratio is typically .

Image of FIG. 5.
FIG. 5.

(Color) Images of the quenching reaction of NATA by NBS. The panels represent the quenching reaction at (a) , (b) , and (c) NBS. The color coding is intended to illustrate the extent of uniformity of the kinetics across the width of the channel. In panel c, the time constant for quenching is comparable to the mixing time. Each vertical scale is per channel.

Image of FIG. 6.
FIG. 6.

(Color) Control reactions testing for protein shearing are shown in (a) and (b). Protein refolding kinetics are shown in Fig. 8. (a) F22W in urea flowing at is diluted by urea flowing at . (b) F22W in buffer flowing at is diluted by buffer flowing at . The fluorescence counts are uniform within error along the flow channel, consistent with the absence of shearing and temperature artifacts. Each trace was acquired on a sample at and utilized a total of approximately of protein. The data have been normalized to a maximum intensity of 1.0.

Image of FIG. 7.
FIG. 7.

(Color) Quenching of NATA fluorescence by NBS monitored by TCSPC detection. The “double-kinetic” experiment features two time axes: corresponds to the nanosecond time-scale excited state decay of NATA, and to that of the microsecond time-scale bimolecular reaction of NBS and NATA. The excited state decay time of NATA does not change as the reaction proceeds, but the population of fluorescing NATA molecules does. A time-resolved decay was acquired for approximately every along the flow channel. Typical count rates were . Only 128 of the 256 decays are shown. The flow rate was .

Image of FIG. 8.
FIG. 8.

Refolding kinetics of horse heart cytochrome in the presence of imidazole collected using TCSPC with an excitation wavelength of and a detection wavelength of . The protein was denatured in Gdn-HCl and diluted tenfold with the microsecond mixing system to a final concentration of Gdn-HCl. The solid line is a biexponential least-squares fit with time constants of and . The superposition of two traces, collected at different scan speeds, is shown. Each trace contains 128 points obtained by integration of the excited-state time-resolved decay of the tryptophan residue. Total collection time for each trace was . The flow rate was , consuming approximately of protein for each trace. Typical count rates were . The vertical scale is in arbitrary units and has been normalized as in Fig. 6.


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: A microchannel solution mixer for studying microsecond protein folding reactions