13 research outputs found
Magnetically Directed Two-Dimensional Crystallization of OmpF Membrane Proteins in Block Copolymers
Get PDFTwo-dimensional (2D) alignment and crystallization of membrane proteins (MPs) is increasingly important in characterizing their three-dimensional (3D) structure, in designing pharmacological agents, and in leveraging MPs for biomimetic devices. Large, highly ordered MP 2D crystals in block copolymer (BCP) matrices are challenging to fabricate, but a facile and scalable technique for aligning and crystallizing MPs in thin-film geometries would rapidly translate into applications. This work introduces a novel method to grow larger and potentially better ordered 2D crystals by performing the crystallization process in the presence of a strong magnetic field. We demonstrate the efficacy of this approach using a β-barrel MP, outer membrane protein F (OmpF), in short-chain polybutadiene-poly(ethylene oxide) (PB-PEO) membranes. Crystals grown in a magnetic field were up to 5 times larger than conventionally grown crystals, and a signal-to-noise (SNR) analysis of diffraction peaks in Fourier transforms of specimens imaged by negative-stain electron microscopy (EM) and cryo-EM showed twice as many high-SNR diffraction peaks, indicating that the magnetic field also improves crystal order
Role of Pore-Lining Residues in Defining the Rate of Water Conduction by Aquaporin-0
Get PDFCompared to other aquaporins (AQPs), lens-specific AQP0 is a poor water channel, and its permeability was reported to be pH-dependent. To date, most water conduction studies on AQP0 were performed on protein expressed in Xenopus oocytes, and the results may therefore also reflect effects introduced by the oocytes themselves. Experiments with purified AQP0 reconstituted into liposomes are challenging because the water permeability of AQP0 is only slightly higher than that of pure lipid bilayers. By reconstituting high amounts of AQP0 and using high concentrations of cholesterol to reduce the permeability of the lipid bilayer, we improved the signal-to-noise ratio of water permeability measurements on AQP0 proteoliposomes. Our measurements show that mutation of two pore-lining tyrosine residues, Tyr-23 and Tyr-149 in sheep AQP0, to the corresponding residues in the high-permeability water channel AQP1 have additive effects and together increase the water permeability of AQP0 40-fold to a level comparable to that of AQP1. Molecular dynamics simulations qualitatively support these experimental findings and suggest that mutation of Tyr-23 changes the pore profile at the gate formed by residue Arg-187
Highly permeable artificial water channels that can self-assemble into two-dimensional arrays
Get PDFBioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(± 0.3) × 10(-14) cm(3)/s or 3.5(± 1.0) × 10(8) water molecules per s, which is in the range of AQPs (3.4 ∼ 40.3 × 10(8) water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10(8) water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼ 10(7) water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼ 2.6 × 10(5) pores per μm(2)) is two orders of magnitude higher than that of CNT membranes (0.1 ∼ 2.5 × 10(3) pores per μm(2)). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays
Energy and techno-economic analysis of bio-based carboxylic acid recovery by adsorption
No full textRecent works have established bio-based carboxylic acids as adaptable precursors to renewable biofuels and chemicals. However, the separation of carboxylic acids is a major energy and cost driver, accounting for 20-40% of the entire processing cost. Improved downstream separation technologies that reduce operating costs compared to conventional approaches are needed, particularly to enable bio-based commodity fuels and chemicals. Here, we combine techno-economic analysis (TEA) and an energy and environmental assessment with experimental results to compare weak-base adsorption (WBA) processes with the conventional strong ion exchange (IX) process for the recovery of the exemplary product, butyric acid. TEA indicates that WBA has the potential to reduce operating expenses from 34% to 6% relative to the selling price of butyric acid ($1.8 kg(-1)). Our energy analysis shows that the WBA process has 12.2-fold energy reduction and 9.2-fold GHG emission reduction compared to the conventional IX process.LPD
PEE–PEO Block Copolymer Exchange Rate between Mixed Micelles Is Detergent and Temperature Activated
No full textPEE–PEO Block Copolymer Exchange Rate between Mixed Micelles Is Detergent and Temperature Activated
No full textWe
examine the kinetics of polymer chain exchange between mixed
block copolymer–detergent micelles, a system relevant to the
synthesis of protein-containing biomimetic membranes. While chain
exchange between block copolymer (BCP) aggregates in water is too
slow to observe, and detergent molecules exchange between micelles
on a time scale of nanoseconds to microseconds, BCP chains exchange
between mixed detergent–polymer micelles on intermediate time
scales of many minutes to a few days. We examine a membrane-protein-relevant,
vesicle-forming, ultrashort polymer, poly(ethyl ethylene)20-poly(ethylene oxide)18 (PEE20–PEO18). PEE20–PEO18 was solubilized
in mixed micelles with the membrane protein compatible nonionic detergent
octyl-β-d-glucoside (OG). Using cryo-TEM and small-angle
neutron scattering (SANS), we demonstrate complete solubilization
of the polymer into micelles. Using time-resolved SANS (TR-SANS),
we provide the first direct evidence that detergents activate BCP
chain exchange and determine kinetic parameters at two detergent concentrations
slightly above the critical micellar concentration (CMC) of OG. We
find that chain exchange increases 2 orders of magnitude when temperature
increases from 35 to 55 °C and that even a 1 mg/mL increase in
OG concentration leads to a noticeable increase in exchange rate.
Our kinetic data are consistent with a single rate-limiting process
rather than the distribution of exchange rates known to exist for
BCPs in the absence of detergent, indicating a different exchange
mechanism than the simple chain escape dominant in single-component
micelles. Using the Arrhenius equation, we determine that at the detergent
concentrations examined the activation energy for polymer chain exchange
is only 2–3 times higher for PEE20PEO18 than for short-chained lipids and that the activation barrier decreases
with increasing OG concentration. On the basis of these results, we
postulate that mixed micelles exchange BCPs through a detergent-mediated
process such as the fusion and fragmentation mechanisms also known
to occur in micellar systems. These findings explain the need for
high detergent concentration and/or temperature to synthesize polymer/protein
membranes. Further, we postulate this is a more general phenomenon
applicable to mixed micelle systems containing amphiphiles with vastly
different solubilities and CMCs differing by many orders of magnitude
Post-Fermentation Recovery of Biobased Carboxylic Acids
No full textCarboxylic
acids are common products produced from the bioconversion
of renewable feedstocks. In these processes the separation of the
acid product from fermentation broth is the most energy and cost intensive
unit operation. Thus, the development of robust, scalable separation
approaches that can be applied to a variety of carboxylates is of
critical importance to the development of processes that utilize carboxylic
acids as platform chemicals. Here we report a batch separation method
that includes cell and particulate removal, cation exchange, activated
carbon treatment, dewatering with a polymer resin, and product recovery.
This method is demonstrated on two unique fermentation broths both
derived from corn stover hydrolysate to separate neat succinic and
propionic acid. For succinic acid, a crystallization yield of 91%
with a product purity of 99.93% was achieved. To our knowledge this
is the highest reported crystallization yield and purity for the recovery
of succinic acid. Additionally, the method requires approximately
50% less energy compared to standard evaporative crystallization approaches.
For propionic acid, neat liquid product was obtained with a distillation
yield of 80% and purity of 98%. These excellent results achieved in
terms of yield and purity for succinic and propionic acid, two acids
with widely different physical properties, from chemically complex
hydrolysate broth demonstrates the effective and robust nature of
this approach
<i>In situ</i> recovery of bio-based carboxylic acids
No full textThe economics of chemical and biological processes is often dominated by the expense of downstream product separations from dilute product streams.</p
