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Microsecond Protein Folding Kinetics

Protein folding events occur on a wide range of timescales.  These are highlighted below with the various techniques used to probe them.  One of the limiting factors in protein folding studies has been access to the sub-millisecond timescale for kinetic measurements. 

Following the lead of the Rousseau, Eaton and Roder groups, we have developed our own laser micromachined microsecond mixer for protein folding studies.  We have developed two mixers with similar designs.  One of them is intended for fluorescence studies and has a dead-time of ~30 microseconds and the other has been adapted for use with SAXS and has a dead-time of ~200 microseconds.  The main limitation for SAXS measurements is the focusing of the x-ray beam.  Although the beam width at half-max is ~35 microns, the SAXS mixer channels need to be wider to make sure that they do not touch the stainless steel edges of the mixing channels.  The continuous-flow SAXS work was done in collaboration with the NIH-funded BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory.  We're very grateful for support from the
NIH (GM23303) and the NSF (MCB0721312) for both the continuous-flow fluorescence and SAXS work at UMass Med.


Both mixer utilize turbulent mixing to achieve mixing.  The timescale of the mixing is reduced by shrinking the dimensions of the mixing chamber to <100 microns.  An overview of turbulent mixing is given in the picture below:




An overview of our mixer design is shown below.  Note that the fluorescence mixer make of PEEK whereas the SAXS mixer is made of stainless steel.



Here's the arrangment of the pumps we use.  Note that the experiment can consume up to 25 mg of protein per kinetic run. We are currently redesigning everything from the ground up to reduce the sample consumption.  We estimate to reduce sample consumption by nearly an order of magnitude when the redesign is complete.


The experimental arrangment of the CF-TCSPC apparatus is shown below.  If the alignment is done properly, there is only a small variation of the excitation intensity as the laser is scanned along the channel (actually, the mixer is moved instead of the laser).  Alignment notes can be found here.





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