Nuclei concentration.

<p>Calculated profiles of nuclei concentrations (pM) versus length (nm), or equivalently time scale, as a function of the bin size. 2 monomers/bin corresponds to 0.47 nm/bin, while 20 monomers/bin corresponds to 4.7 nm/bin.</p

Equations and variables.

<p>Set of equations used to estimate the total number of insulin nuclei, <i>N_n,t</i>, from the available fibril length distribution. The number of measured fibrils per i-th bin, <i>N_fi</i>, Eqs. (1) & (3), were calculated using the Weibull distribution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020072#pone-0020072-g001" target="_blank"><b>Figure 1</b></a>). From the definition of the nucleus, the total number of fibrils, <i>N_f,t</i>, is equivalent to the total number of nuclei, <i>N_n,t</i>, Eq. (4). A description and the units are provided for each variable.</p

Fibril length distribution.

<p>The histogram of frequency versus fibril length summarizes AFM data for 495 insulin fibrils in 36.6 nm/bin for a total of 100 bins. The parameters of this distribution were estimated using distribution-fitting software, EasyFit (MathWave Technologies). The software fitted the data using 60 different distributions and ranked the results based on three different goodness-of-fit tests. The histogram shows the best fit (Kolmogorov-Smirnov statistic, <i>D</i> = 0.0187, Anderson-Darling, <i>A²</i> = 0.323, and Chi-Squared, <i>χ²</i> = 5.113) using the Weibull distribution (line). The probability density function is with values of the parameters: α = 1.7409 and β = 1248.5. (A) Example of a 2D AFM image of insulin fibrils, with measurements: A free-hand curve was drawn on the fibril and two cursors placed at each fibril end. Measurements are in nm. (B) Example of a 3D image, which assisted in detecting individual fibrils.</p

Protein Binding Kinetics in Multimodal Systems: Implications for Protein Separations

In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand’s aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography

Oriented Covalent Immobilization of Antibodies for Measurement of Intermolecular Binding Forces between Zipper-Like Contact Surfaces of Split Inteins

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein <i>trans</i>-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (∼52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski’s equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair

Tranilast Binds to Aβ Monomers and Promotes Aβ Fibrillation

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer’s disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ–tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients