This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Amyloid fibrils are composed of abnormally refolded proteins. AFM has demonstrated its capability to elucidate the in-vitro fibril formation process. The fibril formation of amyloid protein such as Amyloid-? and recombinant IgG light?chain has been reported. The model proposed by Ionescu-Zanetti et al shows that the amyloid proteins form ?-sheet structures to become a single filament in the diameter around 2.4 nm. Two or more filaments can intertwine to form larger size protofilbrils or fibrils directly. This AFM project is related to our other amyloid mass spectrometry projects, and focuses on IgG light-chains purified from patient organs and resuspended in solution, as well as the fibrils taken directly from human organs. AL LC fibrils are suspended in buffer and aliquots are taken at different times for AFM analysis under tapping mode. Our preliminary data showed that the rate of fibril formation is very dependent on the incubation conditions, such as pH and stirring. Fibrils were observed at pH 2 with stirring, but not at pH 5.5 and 7.5. The conformation of the amyloid fibrils purified from patient organs were also measured, and the mass spectral characteristics of each of these and their proteolytic digests were also determined. Different experimental conditions are being tested for IgG light-chain fibril formation. The effects of the presence of glycosaminoglycans, e.g., heparin, heparin sulfate, during the fibril formation are also being explored, using highly sensitive and specific methods that have been developed by the Zaia group. A manuscript that reports the results from AFM and EM imaging of fibril growth was recently published in the Journal of Biological Chemistry.
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