Using NMR to probe transient binding of Aβ1–40 monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19–K28 and a lesser degree of interaction near the C-terminus (L34–G37).

Logic gates with different radixes have been constructed using a biologically active molecule, 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine (DMAPIP-b). Taking advantage of the multiple binding sites of the fluorophore, a series of different molecular logic gates are developed using fluorescence intensities at different wavelengths. The high emission of the molecule is drastically quenched in the presence of Fe(3+). It is regained by the addition of an equivalent amount of F(-). The fluorescence On-Off nature has been used to construct molecular full subtractor and molecular keypad lock system with Boolean logic. A ternary system is generated by considering three defined fluorescence intensities at particular wavelengths. The smooth dependency of emission intensities with analyte concentration is utilized to construct an infinite-valued fuzzy logic system. The fuzzy logic system is further coupled with a neuro-adaptation method to predict more accurately the dependency of molecular intensity on external inputs.

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aβ (TMR-Aβ) to characterize its stability and heterogeneity. Disaggregation of TMR-Aβ amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aβ amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.

Although the interaction of apoE isoforms with amyloid‐β (Aβ) peptides plays a critical role in the progression of Alzheimer's disease, how they interact with each other remains poorly understood. Here, we investigate the molecular mechanism of apoE‐Aβ interactions by comparing the effects of the different domains of apoE on Aβ. The kinetics of aggregation of Aβ1‐42 are delayed dramatically in the presence of substoichiometric, nanomolar concentrations of N‐terminal fragment (NTF), C‐terminal fragment (CTF) and full‐length apoE both in lipid‐free and in lipidated forms. However, interactions between apoE and Aβ as measured by intermolecular Förster resonance energy transfer (FRET) analysis were found to be minimal at t = 0 but to increase in a time‐dependent manner. Thus, apoE must interact with one or more ‘intermediates’ rather than the monomers of Aβ. Kinetics of FRET between full‐length apoE4 labelled with EDANS at position 62 or 139 or 210 or 247 or 276, and tetramethylrhodamine‐labelled Aβ (TMR‐Aβ), further support an involvement of all the three domains of apoE in the interactions. However, the above‐mentioned residues do not appear to form a single pocket in the 3‐dimensional structure of apoE. A competitive binding assay examining the effects of unlabelled fragments or full‐length apoE on the FRET between EDANS‐apoE and TMR‐Aβ show that binding affinity of the full‐length apoE to Aβ is much higher than that of the fragments. Furthermore, apoE4 is found to interact more strongly than apoE3. We hypothesize that high affinity of the apoE‐Aβ interaction is attained due to multivalent binding mediated by multiple interactions between oligomeric Aβ and full‐length apoE.

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