Nature-inspired next generation nanosorters for protein purification

Creating a new class of synthetic membranes with the high selectivity of biological membranes while maintaining large permeation fluxes is the holy grail of membrane science and technology. In Nature, cells have evolved many separators (machines) to select, concentrate and purify water, ions and proteins. In particular, the Nuclear Pore Complex (NPC) is a macromolecular complex that efficiently fractionates proteins between the cell nucleus and cytoplasm in all eukaryotic cells1. Its architecture is well understood and described in the literature,2-5 yet the molecular transport mechanism remains unclear. Transport across the NPC is fast, energy-dependent (to give directionality) and often receptor-mediated. While small molecules pass through the NPCs unchallenged, large macromolecules (\u3e40 kDa) are excluded unless assisted by transport factors collectively termed Karyopherins (Kaps). The translocation of proteins/RNAs occurs through the specific affinity and binding between Kaps and particular nuclear pore complex proteins (nucleoporins) called FG-Nups, which share a degenerate multiple-repeated “Phe-Gly” motif. Because FG-Nups are the major component of the selective gating mechanism, we first investigated the nanomechanical properties of cysteine-modified Nsp1 using the volume force mapping technique of atomic force microscopy (AFM). From single molecule AFM on a sparse Nsp1 surface, we estimated structural parameter as persistence length and contour length. In an attempt to better understand the transport and the selective process under crowding conditions, we then used quartz crystal microbalance with dissipation (QCM-D). Nsp1 and truncated variations of it were immobilized on QCM-D sensors. The binding and unbinding of Kap95, other binding proteins, as well as control proteins, was studied in order to investigate specificity and effect of competitive binding. Finally, we coupled Nsp1 to maleimide functionalized PS-b-PEO membranes and characterized them through X-ray photoelectron spectroscopy. Inspired by Nature, we aim to gain sufficient understanding of the molecular scale engineering principles behind nuclear transport to allow us to design the next generation of synthetic selective nanosorters capable of purifying any protein that we desire. References 1. Grünwald D, Singer RH and Rout M, Nature (2011) 475:333-41 2. Alber F et al., Nature (2007) 450:683-94 3. Alber F et al., Nature (2007) 450:695-701 4. Stuwe T et al., Science (2016) 6226:1148-52 5. Kosinski J et al., Science (2016) 352: 363-6

Evaluating Nuclei Concentration in Amyloid Fibrillation Reactions Using Back-Calculation Approach

Background: In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. Methodology/Principal Findings: Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei Conclusions: Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases

Adsorption and elution of lectins by affinity membranes

Affinity membranes suitable for the purification of lectins were prepared by chemical modifications of a cellulose matrix. As a model protein a lectin obtained by chromatographic techniques from Momordica charantia seeds was mainly used; Peanut agglutinin and Ricinus communis agglutinin were also considered. Different ligands were tested according to the different affinity towards the lectins used. Among the various ligands tested arabinogalactan and N-acetyl-D-galactosamine gave the best performances. The ligand binding reaction onto the epoxy groups of the activated matrices has been optimized with respect to concentration of ligand, temperature and reaction time. The ligand immobilized on the membrane surface is quantified indirectly by measuring the amount of protein bound to the membrane. The kinetics of adsorption and desorption of the purification process has been studied in detail for the different supports. Modified membranes have been used in separation process of lectins with good binding capacity towards the protein of interes

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

Isolating Toxic Insulin Amyloid Reactive Species that Lack β-Sheets and Have Wide pH Stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils

Probing the Nucleus Model for Oligomer Formation during Insulin Amyloid Fibrillogenesis

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers—exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the β-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a β-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy