High Shear Couette Cell Accessory

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This presentation provides a definition of shear and a quick introduction to the notation of fluid mechanics in the context of protein structural analysis. Applied Photophysics has recently introduced the new high shear Couette cell accessory (CCA) which can be used for applications such as to determine the orientation of molecules using linear dichroism or to monitor the change in structure at high shear rates by circular dichroism. To read the full application note, please contact us at info@photophysics.com

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  • Two things that the CCA can be used for, alignment or the investigation of shear induced structural changes
  • Definition of shear and introduction to the notation of fluid mechanics
  • Schematic of the CCA (not to scale). On the right is the top view showing the dimensions
  • Specifications and other features of the CCA
  • Calf thymus DNA at shear rates from 1 to 10,200 s-1. Sample was about 0.12 mg/ml in water. There are two data sets here, low and high shear. Low are averages of 3 repeats, high are averages of two. Baseline was taken as spectrum at zero shear.
  • Top left is the absorbance of about 0.4 mg/ml lysozyme after baseline subtraction measured in the Couette cell at 240/s and 0.5 mm rectangular cells is compared after normalisation for pathlength. Top right is the absorbance of same sample and shear rate measured in the Couette cell in direct, CD and LD modes. Bottom is the CD measured in the Couette cell (red) and 0.5 mm rectangualr cell (cyan) after baseline subtraction and normalisation by absorbanceShear rate used was 240s-1
  • Reason for using a low gap-to-radius ratio. This is mainly to keep the shear rate homogeneous. The Reynolds number describes the shera rate at which the flow is no longer laminar and becomes turbulent (the theoretical value is an upper bound – for real systems the onset of turbulence is always lower).
  • Further explanation of the reason for keeping the gap narrow. In our systems it increases by about 11% from the outer to the inner wall. For other commercially available systems it is much higher.
  • This shows the shear rates at the inner and outer walls at all rotational speeds. The red line is the single value used in our software.
  • Some papers in which shearing has been used for alignment and LD has been used for analysis. The figures show that rods align and vesicles bearing membrane proteins deform in a shear field, giving rise to a non-zero LD signal.
  • Here the LD of DNA (about 0.13 mg/ml) is measured with increasing additions of ethidium bromide from zero to about 28mM. The bottom left figure shows the spectra fro 21 and 28 mM extended to 600 nm. Ethidium bromide intercalates between the DNA bases. This follows work of Tuite and Norden on last slide.
  • Hot off the press from Sandro’s lab. They worked at shear rates from 0 to 3360 s-1, with sugar concentrations from zero to 50% and retinal A concentrations from zero to 50 microM. RetA is the aldehyde of vitamin A, a longish inflexible molecule (lots of conjugated trans double bonds)
  • Do proteins unfold in simple shear? Jaspe and Hagen say not except at very high shear rates (>100000), but the other papers have experimental evidence that suggests otherwise.
  • This followssome work by Ashton et al., Biophysical Journal 96 (2009) 4231-4236, who used Raman spectroscopy to show that changes in the protein conformation occur during shear. They found some distortion of the backbone, but not of the helical structures
  • Shows that we can do temp ramps as well. The signal reduces because of the increase in thermal energy which counters the shearing force and reduces the alignment.
  • And theaudience of course.
  • High Shear Couette Cell Accessory

    1. 1. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Bernard Costello PhD Chirascan High Shear Couette Cell Accessory
    2. 2. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 What does it do? Shears a sample at a set rate: Shearing can be used to generate anisotropy in a sample Used with linear dichroism to determine the orientation of a chromophore relative to the shear axes • e.g. mode of ligand binding to DNA • or of orientation of membrane proteins To investigate the effect of shear on a sample • used with CD or LD, e.g. shear-induced structural changes in proteins
    3. 3. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Shear and shear rate x y Applied stress Almost all fluids in motion are undergoing shear: during pumping, pouring, stirring, shaking, passing down a tube, bearing lubrication, rolling or brushing out, etc.
    4. 4. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Cell dimensions 9.55 mm 9.05 mm Stator Rotor Sample The gap to radius ratio is low (0.25 mm : 4.65 mm 5.4%) to ensure: (1) Homogeneous shear rate (2) High shear rate (3) Low Reynolds number (for circular laminar flow)
    5. 5. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Specifications Chirascan High-Shear Couette Cell Accessory Technical Specifications Design Outer rotating concentric cylinder (Couette) system Shear rate range 0 to 10,200 s-1 Cell pathlength 0.5 mm (i.e. 2 x 0.25 mm) Wavelength Range 180 to 850 nm (Chirascan) or 1150 nm (Chirascan-plus) Sample volume 250 µL Measurement modes LD with simultaneous absorbance, CD with simultaneous absorbance, direct absorbance Purging Fully enclosed for efficient nitrogen purging Temperature control Optional software controlled circulator with close coupled thermocouple Temperature range 5 to 95 C Rectangular cells Standard rectangular cells can be used without removing the accessory
    6. 6. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Difference in absorption between linearly polarised light parallel and perpendicular to orientation direction: ΔA = A‖ - A If the direction of net electron transfer aligns with the orientation direction = No component in perpendicular direction If the direction of net electron transfer is perpendicular to the orientation direction = No component in parallel direction Large molecules or vesicles will align in a shear field Important: shear rate must be laminar and constant in space & time CCA: linear dichroism
    7. 7. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Linear dichroism spectra of DNA LD spectra of DNA at shear rates from 1 to 10,200 s-1 Increasing shear rate
    8. 8. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Lysozyme absorbance and CD: comparison Absorbance: Couette and rectangular cells Absorbance, Couette cell: direct, CD and LD modes CD: Couette and rectangular cells So we can directly obtain the reduced LD: LDR = ALD / A and the CD g-factor: g-factor = ACD / A Comparisons at zero or 240 s-1
    9. 9. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Low gap-to-radius ratio
    10. 10. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Homogenous shear rate
    11. 11. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Shear rate bounds against rotational speed 0 2000 4000 6000 8000 10000 12000 0 1000 2000 3000 4000 5000 Shearrate(1/s) Rotational speed (rpm) Inner wall Outer wall
    12. 12. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Shearing for alignment in the shear field  “Tryptophan orientations in membrane-bound gramicidin and melittin” • Svensson et al, Biochim et Biophys Acta 1808 (2011) 219-228 • LD spectroscopy identifies binding modes for membrane proteins  “Intercalative interactions of ethidium dyes with triplex structures” • Tuite and Norden, Biorg Med Chem 3 (1995) 701-711 • LD spectroscopy identifies binding modes for DNA ligands  Membrane protein orientation • Keller and Textor (work in progress) Zero shear rate (LD = 0) High shear rate (LD ≠ 0) Vesicles with membrane proteins Rods, e.g. DNA
    13. 13. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 DNA with ethidium bromide Increasing [EB] Increasing [EB] Figures show LD (top left) and absorbance (top right) after correction for concentration, of 0.13 mg/ml DNA, at a shear rate of 240 s-1 Ethidium bromide concentration was from zero to about 28 µM
    14. 14. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Retinal A in a vesicle membrane Figures show LD of Retinal A in a vesicle membrane at shear rates from 480 to 3360 s-1 (left), and a contour map of LD as a function of sucrose and RetA concentration. Data are presented with the kind permission of Prof. Sandro Keller and Martin Textor.
    15. 15. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Effect of shear on protein structure “Do protein molecules unfold in simple shear flow?” • Jaspe and Hagen, Biophysical Journal 91 (2006) 3415-3424 • Conflicting reports due to… “heterogeneous velocity conditions” “Shear-induced unfolding of lysozyme monitored in situ” • Ashton et al., Biophysical Journal 96 (2009) 4231-4236 • Raman spectroscopy to detect transitions at 700 s-1 “ -Helix unfolding in simple shear flow” • Bekard et al., Soft Matter 7 (2011) 203-210 • Poly-L-lysine below 715 s-1 in Couette flow using CD “Hydrodynamic effects in proteins” • Szymczak and Ciepak, J. Phys. Cond. Matter 23 (2011) 033102 • von Willebrand factor fibrillation at about 5000 s-1
    16. 16. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Lysozyme 0.3 mg/ml in 95% glycerol Figures show CD (left) and absorbance (right) of 0.3 mg/ml lysozyme in 95% glycerol after baseline correction, at shear rates from zero to 8,000 s-1. The LD was zero throughout.
    17. 17. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 DNA temperature ramp Increasing temperature Temperature ramp for DNA, 20 to 85 C. Measurements made at 240 s-1. The magnitude of the LD decreases because of the increase in thermal energy which counters the alignment force.
    18. 18. PRÄSENTATIONSTITEL, ARIAL 14 International Conference on Chiroptical Spectroscopy, Nashville, June 2013 Acknowledgments Prof. Sandro Keller and Martin Textor of the University of Kaiserslautern Prof. Bengt Norden of Chalmers University of Technology Dr. Alex Drake of Kings College London

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