Advances in Mixer Design and Detection Methods for Kinetics Studies of Macromolecular Folding and Binding on the Microsecond Time Scale

Abstract

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement in terms of temporal resolution and sensitivity. To address this need, we developed a continuous-flow mixing instrument with a dead time of 12 to 27 µs (depending on solution viscosity) and enhanced sensitivity, sufficient for monitoring tryptophan or tyrosine fluorescence changes at fluorophore concentrations as low as 1 µM. Relying on commercially available laser microfabrication services, we obtained an integrated mixer/flow-cell assembly on a quartz chip, based on a cross-channel configuration with channel dimensions and geometry designed to minimize backpressure. By gradually increasing the width of the observation channel downstream from the mixing region, we are able to monitor a reaction progress time window ranging from ~10 µs out to ~3 ms. By combining a solid-state UV laser with a Galvano-mirror scanning strategy, we achieved highly efficient and uniform fluorescence excitation along the flow channel. Examples of applications, including refolding of acid-denatured cytochrome c triggered by a pH jump and binding of a peptide ligand to a PDZ domain, demonstrate the capability of the technique to resolve fluorescence changes down to the 10 µs time regime on modest amounts of reagents.

Keywords: continuous flow; fluorescence; protein folding; protein–ligand interactions; reaction mechanism; turbulent mixing.

Figure A1

Detailed schematic of the layout of Mixer 3, including dimensions (in mm). (A) Overview of the quartz chip. Channel depth is 0.25 mm. (B) Expanded diagram of the mixing region at the intersection of the cross-channels.

Figure 1

Summary of the mixer design and dead-time calibration of three continuous-flow mixing devices. Upper panels show schematics of the mixing regions and mixer/flow-cell assemblies for: (A) Mixer 1, the capillary mixer of Shastry et al. [8], (B) Mixer 2, a simple quartz cross-channel mixing chip (Translume, Inc., Ann Arbor, MI, USA) and (C) Mixer 3, a customized cross-channel mixing chip designed for reduced backpressure and expanded observation window (see Figure A1 in Appendix A for expanded diagram). The lower panels show representative examples of dead-time calibration using the pseudo-first-order NAT-NBS reaction. Typically, the fluorescent reagent (e.g., protein or NAT) is injected into central channel (P), and the second component (e.g., buffer, denaturant, ligand) is injected into the side channels B. The total flow rates in the experiments shown in A, B and C were 1 mL/s, 0.4 mL/s and 0.7 mL/s, respectively. The fluorescence decay curves are color-coded according to NBS concentration: purple for 1 mM, red for 2 mM, brown for 4 mM, green for 8 mM and blue for 16 m. Insets: determination of the second-order rate constant of the NAT-NBS reaction, based on the linear NBS concentration dependence of observed rate constants (determined by single-exponential non-linear least-squares fitting; solid lines).

Figure 2

Optical configuration of our new continuous-flow instrument, using a UV laser in conjunction with a pair of Galvano mirrors to achieve uniform fluorescence excitation along the flow channel of the microfluidic mixing chip (Figure 1B,C).

Figure 3

Calibration of the time axis of Mixer 3. (A) Schematic of the mixing region and conical flow channel (not to scale). The central inlet port (P in Figure 1) is on the left. The right exit is the observation channel. The x-axis is set along the observation channel with the origin on the entrance of it. x₀ is the virtual vertex of the conical observation channel. The exit of the flow channel is located at x = L. The y-axis is set along the cross-section of the channel. The channel widths at the entrance (x = 0) and the exit (x = L) of the flow channel are w₀ and w_L, respectively. (B) Representative profiles of the NAT-NBS quenching reaction. The positions at x = 0.76 mm, 1.1 mm, 2.0 mm, 3.0 mm, 5.0 mm, 10 mm and 23 mm are shown by the lines in brown, red, orange-yellow, green-cyan and blue, respectively. (C) The concentration dependence of fluorescence intensity at representative positions. The data are fitted to Equation (4). (D) The profile of k″t value obtained by curve fitting in panel C. (E) Dependence of distance x on k″t.

Figure 4

Optimization of mixing ratio and flow rate for Mixer 3. (A) Mixing ratio dependence and (B) flow rate dependence of dead time. (C) The NBS concentration dependence of rate constant obtained at several flow rates. The color code is shown in panel C.

Figure 5

Mixing efficiency of Mixer 3 vs. flow rate assayed using potassium iodine quenching of tryptophan fluorescence. Residual fluorescence of NAT (10 mM) upon mixing with 1 mM potassium iodide in the absence (A) and presence (B) of 8 M urea. Color code: 0.4 mL/s (blue), 0.6 mL/s (green), 0.8 mL/s (orange) and 1.0 mL (red). The 95% mixing level is shown by the broken lines. The inlet panels show the time to achieve 95% mixing.

Figure 6

Dead-time calibration performed in the presence of sucrose. NBS is dissolved in 17.9% sucrose, which matches the viscosity of 8.8 M urea (η/η₀ = 1.777). The viscosity of sucrose after 1:10 mixing (16.2%, η/η₀ = 1.663) matches that of 8 M urea.

Figure 7

(A) Kinetics of refolding of acid-denatured cyt c (pH 2, salt-free) triggered by a jump to pH 4.5 (100 mM sodium acetate) recorded using Mixer 1 (green trace, 280 nm excitation) and Mixer 3 (red trace, 266 nm excitation). (B) Kinetics of refolding of cyt c (same conditions as in panel (A)) at different protein concentrations, as indicated.

Figure 8

Binding kinetics of peptides from the C-terminus of CFTR to the first PDZ domain of NHERF1. (A) Kinetic traces of CFTR₁₀ binding to PDZ1 of NHRF1. (B) Rate constants of the binding reaction for CFTR₁₀ (blue) and CFTR₆ (green). The line represents a hyperbolic fit of the rate constant vs. ligand concentration.