Sing the dialysis method. Preparations of Ab42CC protofibrils are stable towards dissociation when diluted with buffer, while smaller Ab42CC oligomers may dissociate into monomeric species upon dilution (A. Sandberg and A. Dubnovitsky, unpublished). Protofibril preparations are also stable towards dissociation for months at room temperature.NaCl and sample preparation for AFM in air requires removal of salts. This may be achieved by diluting concentrated protofibril solutions in deionized water before loading them onto mica. However, with such a procedure only smaller Ab42CC protofibrils appear to bind the mica. Therefore, special care was taken to minimize exposure to low-salt environment. Purified protofibrils were diluted in buffer, loaded onto freshly cleaved mica, and briefly washed with water. Prepared in this way, Ab42CC protofibrils appear in AFM as unbranched rod-like structures with a well defined order 1113-59-3 average height of 3.1 (60.16) nm and typical lengths in the range 50 to 220 nm (Fig. 1A ). The largest protofibril is more than 600 nm long (Fig. S1). The protofibril width measured here (AFM z-height) is smaller than widths of wild type Ab protofibrils [23,24] and Ab42CC protofibrils [16] observed in electron microscopy with uranyl acetate negative staining (6 to 10 nm), but a 3.1 nm height was also reported in a detailed AFM analysis of protofibrils of wild type Ab40 [25]. High-resolution scans of single protofibril particles reveal a smooth surface without distinguishing features (Fig. 1C). The shape and size of Ab42CC protofibrils are identical to those of wild type Ab42 protofibrils identified in aggregation reaction mixtures of 100 mM peptide stored at room temperature without shaking (Fig. 1B , Fig. S2). The morphology of Ab42CC and Ab42 protofibrils are both very different from the characteristic helical appearance of Ab40 or Ab42 fibrils imaged under the same conditions (Fig. 1E ). (The dimension and morphology of the Ab40 fibril are such that it may be envisioned as a double helix of 3.1 nm filaments.) Short Ab42CC protofibrils appear to be straight in AFM, but smooth curvatures can be observed with increasing particle length. The precise persistence length of the rods was, however, not determined. If protofibrils bound to mica are extensively washed with water, more sharp kinks appear together with multiple brakes in long protofibrils. We characterized the protofibril length distributions in two different samples of Ab42CC protofibrils (Fig. 2). One sample was washed only very briefly with water, as above, while the other was washed extensively several times. Extensive washing with water disrupts the protofibrils (on mica) and many particles become shorter than 100 nm (Fig. 2). Interestingly, the two size distributions show a similar fine structure (or periodicity) indicating that protofibril disruption involves dissociation of discrete oligomeric building blocks.Protofibril size distribution in solutionThe size distribution of Ab42CC protofibrils in solution was analyzed by non-invasive analytical ultracentrifugation (AUC). Data analysis assuming a continuous c(s) distribution model indicates a distribution of large particles between 2 and 30 S (Fig. 3). A size distribution centered at s20, w = 18 S with a best-fit frictional ratio f/f0 of 2.7 to 3.1 suggests the presence of long 374913-63-0 rodlike particles. For comparison, f/f0 = 2.3 and s20, w = 7.6 for human fibrinogen (MW = 330,000 Da), and f/f0 = 3.6 and s20, w = 6.4 for myosi.Sing the dialysis method. Preparations of Ab42CC protofibrils are stable towards dissociation when diluted with buffer, while smaller Ab42CC oligomers may dissociate into monomeric species upon dilution (A. Sandberg and A. Dubnovitsky, unpublished). Protofibril preparations are also stable towards dissociation for months at room temperature.NaCl and sample preparation for AFM in air requires removal of salts. This may be achieved by diluting concentrated protofibril solutions in deionized water before loading them onto mica. However, with such a procedure only smaller Ab42CC protofibrils appear to bind the mica. Therefore, special care was taken to minimize exposure to low-salt environment. Purified protofibrils were diluted in buffer, loaded onto freshly cleaved mica, and briefly washed with water. Prepared in this way, Ab42CC protofibrils appear in AFM as unbranched rod-like structures with a well defined average height of 3.1 (60.16) nm and typical lengths in the range 50 to 220 nm (Fig. 1A ). The largest protofibril is more than 600 nm long (Fig. S1). The protofibril width measured here (AFM z-height) is smaller than widths of wild type Ab protofibrils [23,24] and Ab42CC protofibrils [16] observed in electron microscopy with uranyl acetate negative staining (6 to 10 nm), but a 3.1 nm height was also reported in a detailed AFM analysis of protofibrils of wild type Ab40 [25]. High-resolution scans of single protofibril particles reveal a smooth surface without distinguishing features (Fig. 1C). The shape and size of Ab42CC protofibrils are identical to those of wild type Ab42 protofibrils identified in aggregation reaction mixtures of 100 mM peptide stored at room temperature without shaking (Fig. 1B , Fig. S2). The morphology of Ab42CC and Ab42 protofibrils are both very different from the characteristic helical appearance of Ab40 or Ab42 fibrils imaged under the same conditions (Fig. 1E ). (The dimension and morphology of the Ab40 fibril are such that it may be envisioned as a double helix of 3.1 nm filaments.) Short Ab42CC protofibrils appear to be straight in AFM, but smooth curvatures can be observed with increasing particle length. The precise persistence length of the rods was, however, not determined. If protofibrils bound to mica are extensively washed with water, more sharp kinks appear together with multiple brakes in long protofibrils. We characterized the protofibril length distributions in two different samples of Ab42CC protofibrils (Fig. 2). One sample was washed only very briefly with water, as above, while the other was washed extensively several times. Extensive washing with water disrupts the protofibrils (on mica) and many particles become shorter than 100 nm (Fig. 2). Interestingly, the two size distributions show a similar fine structure (or periodicity) indicating that protofibril disruption involves dissociation of discrete oligomeric building blocks.Protofibril size distribution in solutionThe size distribution of Ab42CC protofibrils in solution was analyzed by non-invasive analytical ultracentrifugation (AUC). Data analysis assuming a continuous c(s) distribution model indicates a distribution of large particles between 2 and 30 S (Fig. 3). A size distribution centered at s20, w = 18 S with a best-fit frictional ratio f/f0 of 2.7 to 3.1 suggests the presence of long rodlike particles. For comparison, f/f0 = 2.3 and s20, w = 7.6 for human fibrinogen (MW = 330,000 Da), and f/f0 = 3.6 and s20, w = 6.4 for myosi.
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