Abstract
Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-time-resolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.
Highlights
Time-resolved studies of structural changes in biological macromolecules are of fundamental importance in understanding their biological function
The micro-Small-angle X-ray scattering (SAXS) continuous-flow approach was applied to the folding of cytochrome c, a well studied model system known to exhibit kinetics in the sub-millisecond time regime (Chan et al, 1997; Shastry & Roder, 1998)
Previous continuous-flow SAXS and fluorescence studies have indicated that the transition from a random-coil-like state to the native state occurs with a sub-100 ms kinetic step followed by a 650 ms step (Chan et al, 1997; Shastry & Roder, 1998)
Summary
Time-resolved studies of structural changes in biological macromolecules are of fundamental importance in understanding their biological function. Better time resolution can be achieved using a smaller X-ray beam focal spot that would permit using narrower mixer channels Another limitation was the low duty cycle ($ 10%) dictated by the slow readout of the CCD-based area detectors used in these earlier experiments. A channel width smaller than 100 mm is needed to achieve turbulent mixing with minimum dead-times in the 50 ms time range as demonstrated with fluorescence studies (Bilsel et al, 2005) This width is sufficient to avoid scattering from the tails of the intensity distribution of the X-ray microbeam. At the flow rates currently used ($ 120 ms mmÀ1), the beam dimension parallel to the flow direction is negligible in its effect on time resolution. (It would become an issue only if the mixing time was faster than 15 ms.)
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