PEG 8K Research Articles

Yield enhancement and phase behaviors of cyclic peptide drug crystallization in the presence of polyethylene glycol additive

Crystallization of peptide drugs typically requires a lengthy period to attain reasonable yields due to slow crystallization kinetics of macromolecules. Herein we investigated the use of polyethylene glycol (8 kDa) (PEG 8k) as an additive to enhance the yield of salting-out crystallization of cyclic peptide drug vancomycin (Van). Our preliminary study showed the addition of PEG 8k increased the 24-h yield from 46 wt% to 70 wt% attributed to the polymer’s volume-exclusion effects. PEG 8k addition, however, resulted in modified Van crystal habits, thereby phase behavior study was performed at different pH, salt, polymer, and Van concentrations. The phase behavior study showed PEG 8k and salt concentrations must be meticulously determined to produce stable Van crystals because their excessive concentrations resulted in heavy precipitates. Different crystal habits (e.g., acicular, blade-like) were produced with PEG 8k additive depending on the pH, distinct from octahedral crystals produced without additive. Bulk crystallization with PEG 8k additive resulted in platy Van crystals with higher crystallinity, lower thermal stability and purity, but with similar dissolution rates compared to octahedral crystals prepared without PEG 8k. PEG 8k addition had catalytic effects on the salting-out yield whereby lower salt concentration was needed. In short, PEG 8k addition enhanced the yield without compromising Van crystals’ qualities.

Molecular Compressive Force Sensor for Mapping Forces at the Cell-Substrate Interface.

Mechanical forces are crucial for biological processes such as T cell antigen recognition. A suite of molecular tension probes to measure pulling forces have been reported over the past decade; however, there are no reports of molecular probes for measuring compressive forces, representing a gap in the current mechanobiology toolbox. To address this gap, we report a molecular compression reporter using pseudostable hairpins (M-CRUSH). The design principle was based on a pseudostable DNA structure that folds in response to an external compressive force. We created a library of DNA stem-loop hairpins with varying thermodynamic stability, and then used Förster Resonance Energy Transfer (FRET) to quantify hairpin folding stability as a function of temperature and crowding. We identified an optimal pseudostable DNA hairpin highly sensitive to molecular crowding that displayed a shift in melting temperature (Tm) of 7 °C in response to a PEG crowding agent. When immobilized on surfaces, this optimized DNA hairpin showed a 29 ± 6% increase in FRET index in response to 25% w/w PEG 8K. As a proof-of-concept demonstration, we employed M-CRUSH to map the compressive forces generated by primary naïve T cells. We noted dynamic compressive forces that were highly sensitive to antigen presentation and coreceptor engagement. Importantly, mechanical forces are generated by cytoskeletal protrusions caused by acto-myosin activity. This was confirmed by treating cells with cytoskeletal inhibitors, which resulted in a lower FRET response when compared to untreated cells. Furthermore, we showed that M-CRUSH signal is dependent on probe density with greater density probes showing enhanced signal. Finally, we demonstrated that M-CRUSH probes are modular and can be applied to different cell types by displaying a compressive signal observed under human platelets. M-CRUSH offers a powerful tool to complement tension sensors and map out compressive forces in living systems.

RNA sequence and structure control assembly and function of RNA condensates.

Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.

On the exfoliating polymeric cellular dosage forms for immediate drug release

The most prevalent pharmaceutical dosage forms at present—the oral immediate-release tablets and capsules—are granular solids. Though effective in releasing drug rapidly, development and manufacture of such dosage forms are fraught with difficulties inherent to particulate processing. Predictable dosage form manufacture could be achieved by liquid-based processing, but cast solid dosage forms are not suitable for immediate drug release due to their resistance to fluid percolation. To overcome this limitation, we have recently introduced cellular dosage forms that can be readily prepared from polymeric melts. It has been shown that open-cell structures comprising polyethylene glycol 8000 (PEG 8k) excipient and a drug exfoliate upon immersion in a dissolution medium. The drug is then released rapidly due to the large specific surface area of the exfoliations. In this work, we vary the molecular weight of the PEG excipient and investigate its effect on the drug release kinetics of structures with predominantly open-cell topology. We demonstrate that the exfoliation rate decreases substantially if the excipient molecular weight is increased from 12 to 100kg/mol, which causes the drug dissolution time to increase by more than a factor of ten. A model is then developed to elucidate the exfoliation behavior of cellular structures. Diverse transport processes are considered: percolation due to capillarity, diffusion of dissolution medium through the cell walls, and viscous flow of the saturated excipient. It is found that the lower exfoliation rate and the longer dissolution time of the dosage forms with higher excipient molecular weight are primarily due to the greater viscosity of the cell walls after fluid penetration.

Differential Effects of Ions, Molecular Crowding, and Solution DNA Density on the Damage Search Mechanisms of hOGG1 and hUNG

The rarity of DNA damage within the human genome results in DNA repair enzymes interacting primarily with undamaged DNA. Interestingly, despite the fact that different human DNA glycosylases have similar search and repair requirements, these enzymes have evolved different ways of rapidly distinguishing between their target lesion and undamaged bases. In the UDG superfamily, human uracil DNA glycosylase (hUNG) makes extensive contact with the phosphate backbone of undamaged DNA with negligible distortion of the DNA helix. In contrast, human 8-oxo-guanine DNA glycosylase (hOGG1) of the HhH-GPD superfamily displaces undamaged guanine bases from the helix and induces a 70o bend angle in the DNA. Here we investigate how these different non-specific DNA complexes affect the ability of hUNG and hOGG1 to execute efficient damage search and repair within the context of duplex DNA. We find that the specificity of hUNG for uracilitated DNA is enhanced under physiological conditions because non-specific DNA binding is weakened by high ion concentrations, while specific binding of uracil in DNA is not. In contrast, non-specific DNA binding by hOGG1 is relatively insensitive to high monovalent ion concentrations. We report on the consequences of this distinct response to high ionic strength for facilitated diffusion along DNA. Specifically, we show that the presence of the artificial crowding agent PEG 8K negates the deleterious effects of high salt and that facilitated diffusion along short distances persists despite the presence of potentially inhibitory excess nonspecific DNA. These results establish an in vitro model for how specific features of the cellular environment synergistically facilitate efficient DNA repair.

Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16.

The gene product of A1887 from Ralstonia eutropha (ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min(-1) and 5 × 10(6) s(-1), respectively. The ReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 M HEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 129.52, c = 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.

Crystallization and preliminary X-ray diffraction analysis of Trap1 complexed with Hsp90 inhibitors.

Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293 K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1-PU-H71 (2.7 Å) and Trap1-BIIB-021 (3.1 Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 69.2, c = 252.5 Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.

Crystallization and preliminary X-ray diffraction studies of cyanuric acid hydrolase from Azorhizobium caulinodans.

Cyanuric acid is synthesized industrially and forms during the microbial metabolism of s-triazine herbicides. Cyanuric acid is metabolized by some microorganisms via cyanuric acid hydrolase (CAH), which opens the s-triazine ring as a prelude to further metabolism. CAH is a member of the rare cyanuric acid hydrolase/barbiturase family. Here, the crystallization and preliminary X-ray diffraction analysis of CAH from Azorhizobium caulinodans are reported. CAH was cocrystallized with barbituric acid, a close analog of cyanuric acid that is a tight-binding competitive inhibitor. Crystals suitable for X-ray diffraction experiments were grown in conditions containing PEG 8K or magnesium sulfate as precipitants. An X-ray diffraction data set was collected from CAH-barbituric acid crystals to 2.7 Å resolution. The crystals were found to belong to space group I4₁22, with unit-cell parameters a = b = 237.9, c = 105.3 Å, α = β = γ = 90°.

Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus.

Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2(1)2(1)2 and diffracts up to 2.1 Å and the RdRp-Lu(3+) derivative co-crystals belong to the C222(1) space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses.

Engineering extruded collagen fibers for biomedical applications

AbstractExtruded collagen fibers constitute a promising biomimetic scaffold for tissue engineering applications. In this study, we compared the structural, thermal, and mechanical properties of fibers produced from either NaCl or poly(ethylene glycol) with a number‐average molecular weight of 8000 (PEG 8K), the only two coagents that have been used in the fabrication process. As novel, we report the fabrication of fibers with properties similar to native or synthetic fibers using other coagents. NaCl derived fibers were characterized by higher thermal stability (p < 0.026), stress (p < 0.001), and modulus (p < 0.0025) values than PEG 8K, whereas the latter yielded more extendable fibers (p < 0.012). Poly(ethylene glycol)s with number‐average molecular weights of 200 and 1000 produced fibers with similar mechanical properties (p > 0.05) that were thinner (p < 0.033), stiffer (p < 0.022), and less extendable (p < 0.0002) than those of PEG 8K. Poly(vinyl alcohol) (PVA) with a number‐average average molecular weight of 9–10,000 and PEG 8K yielded fibers with similar diameters and stress‐at‐break values (p > 0.05); however, the poly(ethylene glycol) derived fibers were more extendable (p < 0.0003), whereas the PVA fibers were stiffer (p < 0.029). Gum‐arabic‐ and soluble‐starch‐derived fibers were of similar tensile strength, extendibility, and stiffness (p > 0.05). In this in vitro study, the thickest (p < 0.011) and the weakest (p < 0.0066) fibers were produced in the presence of sodium sulfate. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Crystallization and preliminary crystallographic study of the yeastMalassezia sympodialisallergen Mala s 1

The opportunistic yeast Malassezia sympodialis can act as an allergen and elicit specific IgE- and T-cell reactivity in patients with atopic eczema. The first identified major allergen from M. sympodialis, Mala s 1, is present on the cell surface of the yeast. Recombinant Mala s 1 was expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Mala s 1 were obtained in 25% PEG 8K, 0.2 M (NH4)2SO4. Crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 44.4, b = 163.7, c = 50.6 A, and diffract to 1.35 A resolution.

Crystals of family 11 xylanase II from Trichoderma longibrachiatum that diffract to atomic resolution.

Xylanases catalyse the cleavage of various forms of xylan. A new crystal form of xylanase II from the fungus Trichoderma longibrachiatum that diffracts to better than 1 A resolution was grown from 12% PEG 8K, 0.1 M Tris pH 8.5, 0.2 M CaCl(2) with the addition of 2% glycerol to overcome crystal twinning. The crystals grow in a body-centered orthorhombic Bravais lattice, with unit-cell parameters a = 66.78, b = 67.94, c = 79.18 A. The solvent content is 42% with one molecule per asymmetric unit. Molecular-replacement analysis reveals the space group to be I222. This atomic resolution structure will provide important insights that will lead to a better understanding of the enzymatic mechanism of the family 11 xylanases.

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P21 space group with unit cell parameters a=136.06, b=206.59, c=227.39 Å, β=105.03°. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P212121 with unit cell parameters of a=44.01, b=44.30, c=122.47 Å. The diffraction data of the complex were collected up to a resolution of 2.4 Å using a synchrotron-radiation source under cryogenic condition.

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P2 1 space group with unit cell parameters a=136.06, b=206.59, c=227.39 Å, β=105.03°. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P2 12 12 1 with unit cell parameters of a=44.01, b=44.30, c=122.47 Å. The diffraction data of the complex were collected up to a resolution of 2.4 Å using a synchrotron-radiation source under cryogenic condition.

Bacillus subtilis division protein DivIVA - screen for stable oligomer state conditions.

The cell division protein DivIVA is predicted to be a coiled-coil, tropomyosin-like protein, that self-associates both in vivo and in vitro into oligomers of up to 10-12 monomers. A simple and quick screen for conditions supporting the stable oligomer structure has been developed revealing that DivIVA forms a homogeneous oligomer in the presence of PEGs (PEG 4 K or PEG 8K and PEG 1K).

Crystallographic study of a naturally occurring trans-splicing intein from Synechocystis sp. PCC 6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been purified and crystallized using PEG 8K as precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.5, c = 70.2 A. It has one molecule per asymmetric unit and diffracts to beyond 2.9 A under cryoconditions (110 K) using a Cu rotating-anode X-ray generator in-house.

Crystallization and preliminary X-ray analysis of the Escherichia coli UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (MurF).

Crystals of the Escherichia coli UDP-MurNAc-tripeptide D-Ala-D-Ala-adding protein (MurF), which catalyzes the formation of the last metabolite of the bacterial cell-wall building block, have been grown in hanging-drop vapor-diffusion trials using PEG 8K as a precipitating agent. The crystals belong to hexagonal space group P6(1) or P6(5), with unit-cell dimensions a = b = 74, c = 425 A. The asymmetric unit contains two molecules, with a crystal volume per protein mass (V(m)) of 3.4 A(3) Da(-1) and a solvent content of about 64% by volume. A native data set to 2.8 A resolution has been obtained from a frozen crystal using a synchrotron X-ray source.

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles.

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.