Advances in turbulent mixing techniques to study microsecond protein folding reactions

Abstract

Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically-denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10’s of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer (FRET), Circular Dichroism (CD), Small-Angle X-ray Scattering (SAXS), Hydrogen Exchange (HX) followed by Mass Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR), Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy (FTIR), has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst-phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, that are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics

“…If users cannot slow their experiments enough to conduct a hand-mixed experiment, they can make comparisons to experiments conducted by rapid-quench or other fast reaction methodologies, such as stopped-flow [27, 39, 40], or continuous flow-associated techniques [32, 41]. Alternatively, other test reactions are available such as the base-catalyzed hydrolysis of benzylidene malanonitrile (BMN), and can be recommended by KinTek upon inquiry through their website.…”

Frustration and folding of a TIM barrel protein

“…We provide connections between the off-pathway intermediate and one on-pathway rate-determining intermediate detected by experiment with corresponding meta-stable states in the major folding channel found in the simulations. folding events in the microsecond time range and have allowed us to examine the earliest events in the folding of a candidate TIM barrel protein (13). In the present study, pair-wise distance measurements from time-resolved Förster resonance energy transfer (trFRET) and global size and shape measurements from small-angle X-ray scattering (SAXS), combined with coarsegrained computer simulations, are employed to probe the earliest events in the folding of the Sulfolobus solfataricus indole-3glycerol phosphate synthase (SsIGPS) TIM barrel.…”

Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging

“…Rapid mixing of reagents is certainly the most popular principle to retrieve kinetic information. It can be implemented in bulk using a stopped‐flow apparatus or some other rapid mixing devices, or it can be achieved at the surface of a solid‐state sensor . Usually one analyzes the response to a concentration step in the time domain.…”