Experiments on alignment of amyloid fibrils
Overview
Amyloid fibrils are insoluble protein aggregates that have been associated with a range of diseases including BSE, Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob Disease (CJD). They are formed by a range of different proteins and peptides, and are characterised by common structural features, whereby stacks of parallel peptide strands running perpendicular to the fibril axis hydrogen-bond to form extended beta-sheets (above, left). It has been suggested that a number of these sheets then stack across the fibril axis, to form higher-order structures which ultimately give rise to fibrils a few nanometres in diameter, and up to a few microns in length (above, right).
Apparatus
I have constructed flow cells based on two different geometries to align these fibrils; a parallel-plate continuous shear cell, where a pure shear field induces alignment, and a number of elongational flow cells, where the fibrils align due to a non-uniform flow rate along the length of the cell. The cells have inter-changeable windows to allow simultaneous analysis by birefringence, wide-angle x-ray scattering, or polarised vibrational spectroscopy. Flow-alignment allows analysis of fibril structure without the need to dry the sample.
Birefringence
The above images show a round drop of amyloid fibril suspension shear-aligned in the parallel plate shear cell. Both images were photographed through cross-polars. The image on the left shows the characteristic "maltese cross" pattern commonly seen in polymer spherulites or liquid crystalline defects with circular symmetry (see, for example, this flash animation ). Of course, this image shows circular ordering on a macroscopic rather than microscopic length scale (the drop in the above picture is approximately 1cm in diameter). The image on the right used an additional retardation plate, confirming that alignment is tangential rather than radial, ie, parallel to flow direction.
Wide-Angle X-ray scattering
The characteristic wide-angle x-ray diffraction pattern from the "cross-beta" structure described above is two pairs of arcs. The two diffraction patterns shown above were taken from samples with fibrils oriented vertically. The outer pair of arcs, above and below the beam centre, correspond to the spacing between beta-strands of approximately 4.7 Angstroms. The pair further in, to the left and right of the beam centre, correspond to the spacing between beta-sheets of around 8-10 Angstroms. Any reflections even further in arise either from inter-protofilament scattering, or from scattering by the beam-stop.
The image on the left was obtained from a dry aligned stalk, using Kodak autoradiography film, on the BSS group x-ray beamline at the Cavendish laboratory, Cambridge. The image on the right was obtained from a flow-aligned sample after background subtraction, on beamline 14.1 at Daresbury synchrotron source.
Microscopy
The extensional flow-cell can be used to deposit aligned amyloid fibrils onto a mica substrate, for imaging using atomic force microscopy (AFM). In the above image, the flow direction was approximately 20 degrees to the left of vertical, relative to the image.
Raman Spectroscopy
I am currently collaborating with the research group of Professor Igor Lednev, in SUNY, Albany NY, on the possible use of Raman spectroscopy on these systems.
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