Atomic force microscopes (AFMs) are ubiquitous in research laboratories and have recently been priced for use in teaching laboratories. Here we review several AFM platforms (Dimension 3000 by Digital Instruments, EasyScan2 by Nanosurf, ezAFM by Nanomagnetics, and TKAFM by Thorlabs) and describe various biophysical experiments that could be done in the teaching laboratory using these instruments. In particular, we focus on experiments that image biological materials and quantify biophysical parameters: 1) imaging cells to determine membrane tension, 2) imaging microtubules to determine their persistence length, 3) imaging the random walk of DNA molecules to determine their contour length, and 4) imaging stretched DNA molecules to measure the tensional force.2

factor. One of the possible dysfunctional fates of apoA-I is represented by protein aggregation and amyloid fibril formation; as suggested by high incidence of apoA-I amyloid deposits in atherosclerotic lesions. Recently it has been reported that upon oxidation of its three native methionines, apoA-I can produce amyloid fibrils in vitro. The aim of our study was to establish the contribution of oxidation of specific methionines to apoA-I amyloid fibril formation. Single amino acid apoA-I mutants were produced, in which each of the three native methionines (M86, M112, and M148) were replaced by leucines. After oxidation by H 2 O 2 and removal of H 2 O 2 by dialysis, the protein samples were incubated at 37 C and pH 6.0 to elicit protein aggregation. Amyloid fibril formation was evaluated by measuring the reactivity of the samples towards the fluorescent dye Thioflavin T (ThT). Oxidized wild-type apoA-I (WT-apoA-I) readily formed solid aggregates. ThT fluorescence developed with rapid kinetics and reached maximum intensity at 24h. Upon oxidation and incubation in similar conditions, ThT kinetics were distinct for different single M to L apoA-I mutants, suggesting that the three methionines do not equally contributes, upon oxidation, to protein destabilization and the process of amyloid fibril formation. In particular, ThT kinetics of M112L-and WT-apoA-I were not significantly different (T 1/2 @ 4 and 3 h, respectively). In contrast, ThT kinetics were significantly slower for both M86L-apoA-I and M148L-apoA-I (T 1/2 @ 30 h and > 48 h, respectively). These results indicate that oxidation of M86 and M148 is largely responsible for apoA-I destabilization and amyloid fibril formation. 2380-Pos Board B72Use of FCS to Study Protein Denaturation and Aggregation Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying the diffusive dynamics of fluorophore-tagged biomolecules and thereby drawing inferences about their conformational states and transitions. We have employed FCS to investigate the denaturation and aggregation pathways of bovine immunoglobulin (IgG), a multi-subunit all beta-sheet Y-shaped protein prone to aggregation. Each upper arm of the Y consists of a light and a heavy chain connected by cysteine-shy;cysteine disulphide bonds, and the two heavy chains are joined at the bend by more disulphides. While majority of proteins show continuous increase of hydrodynamic radius (Rg) when presented with increasing denaturant concentration, IgG exhibits strikingly different behavior under similar conditions. Its Rg initially decreases with increasing concentration of GdnHCl (from 0 to 3 M) and then goes through two pronounced maxima at 4 M and 7 M [GdnHCl]. We propose that the initial contraction is related to sheet-to-helix transition at the flexible bends of the Y-arms of the protein. The first maximum of Rg is attributed to electrostatic repulsions and their screening by Gdnþ and Cl-ions; the second is shown to arise from disulphide-mediated effects. Previous studies have proposed that IgG a...

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Although people are generally interested in how the brain functions, neuroscience education is hampered by a lack of low cost and engaging teaching materials. To address this, we developed an open-source lipid bilayer amplifier which is appropriate for use in introductory courses in biophysics or neurosciences. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. This device can be used in simple experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionophores like gramicidin A. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.