In vitro evolution of a hyperstable Gbeta1 variant

An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.     

薄层扫描法测定产妇定胶囊中盐酸水苏碱的含量

为建立一种控制产妇定胶囊质量的方法,本文采用薄层扫描法测定处方中君药益母草中所含的主要有效成分盐酸水苏碱的含量.结果表明盐酸水苏碱在3.090～30.900μg范围内线性关系良好,平均回收率为99.06%,RSD=2.57%(n=5).该方法灵敏、准确,分辨率高,可用于产妇定胶囊的质量控制.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Synthesis and application of thiol‐functionalized magnetic nanoparticles for studying interactions of epirubicin hydrochloride with bovine serum albumin by fluorescence spectrometry

A novel method was developed for studying the interaction between epirubicin hydrochloride (EPI) and bovine serum albumin (BSA) by fluorescence spectrometry. Fe₃O₄ magnetic nanoparticles (MNPs) synthesized and functionalized with thiol group were employed for the immobilization and separation of target BSA in reaction solutions. The concentrations of the non‐immobilized BSA and unbound EPI were obtained separately by fluorescence spectrometry. The binding constants (K _a ) and number of binding sites (n ) of EPI with BSA were calculated. In this study, the K _a value was 5.05 × 10⁵ L mol^?1, suggesting a strong binding of EPI to BSA, and the n value was 1.15. The effects of common metal ions on K _a of EPI with BSA were also investigated, and the results showed there was clearly bindings between the metal ions and BSA. The precise binding sites of EPI on BSA were determined as being in site I from the competitive displacement experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t_1/2  ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor a 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating that it serves as the sensor.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

Characterization of antioxidant enzymes and peroxisomes of olive (Olea europaea L.) fruits

The presence of peroxisomes in olive (Olea europaea L.) fruits and different antioxidant enzymes occurring in this plant tissue is reported for the first time. Ultrastructural analysis showed that olive cells were characterized by the presence of large vacuoles and lipid drops. Plastids, mitochondria and peroxisomes were placed near the cell wall, showing some type of association with it. Olive fruit peroxisomes were purified by sucrose density-gradient centrifugation, and catalase, glutathione reductase and ascorbate peroxidase were found in peroxisomes. In olive fruit tissue the presence of a battery of antioxidant enzymes was demonstrated, including catalase, four superoxide dismutase isozymes (mainly an Fe-SOD plus 2 Cu,Zn-SOD and a Mn-SOD), all the enzymes of the ascorbate–glutathione cycle, reduced and oxidized glutathione, ascorbate, and four NADPH-recycling dehydrogenases. The knowledge of the full composition of antioxidants (enzymatic and non-enzymatic) in olive fruits is crucial to be able to understand the processes regulating the antioxidant composition of olive oil.     

The Use of Phosphite‐Type Ligands in the Ir‐Catalyzed Asymmetric Hydrogenation of Heterocyclic Compounds

A series of chiral phosphite‐type ligands was tested in asymmetric Ir‐catalyzed hydrogenation of quinolines and 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole. Hydrogenation of quinaldine hydrochloride provided superior enantioselectivity up to 65% ee compared to quinaldine free base. The ligands were tested for the first time in the asymmetric Ir‐Ircatalyzed hydrogenation of 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole yielding the antidepressant drug, pirlindole. Chirality 26:56–60, 2013. © 2013 Wiley Periodicals, Inc.