Binding Behaviors for Different Types of DNA G-Quadruplexes: Enantiomers of [Ru(bpy)2(L)](2+) (L=dppz, dppz-idzo).

Polymorphic DNA G-quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)2 (L)](2+) (L=dipyrido[3,2-a:2',3'-c]phenazine, dppz-10,11-imidazolone; bpy=2,2'-bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G-quadruplexes. These studies provide a striking example of chiral-selective recognition of DNA G-quadruplexes. As for antiparallel (tel-Na(+)) basket G-quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B-DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel-K(+)) G-quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G-quadruplex. Whereas both Δ and Λ isomers bind to parallel G-quadruplexes with comparable affinity, no appreciable stereoselective G-quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G-quadruplex binding is not only related to G-quadruplex topology, but also to the sequence and the loop constitution.

Molecular "light switch" [Ru(phen)2dppzidzo](2+) monitoring the aggregation of tau.

Monitoring the aggregation of the tau protein is a key protocol for elucidating the pathogenic mechanism of Alzheimer's disease. In the present article, [Ru(phen)2dppzidzo](2+), a "light switch" ruthenium(ii) complex, was presented as a new monitoring probe for the aggregation of a tau R3 peptide, the third repeat unit of the tau microtubule-binding domain. Having little impact on the aggregation process, large fixed Stokes shift and small background luminescence made the complex a better probe for monitoring the aggregation process and quantitatively detecting tau filaments compared to thioflavin S, a commonly used fluorescent dye for staining neurofibrillary tangles and monitoring tau aggregation. Furthermore, a long luminescence lifetime of this complex could also expand its potential usage in the detection of tau filaments in the presence of short-lived fluorescent backgrounds.

A new fluorescence "switch on" assay for heparin detection by using a functional ruthenium polypyridyl complex.

In the present study, a new strategy for heparin detection and quantification in biological media, such as fetal bovine serum (FBS), is developed by monitoring the emission change of a functional ruthenium polypyridyl complex ([Ru(phen)(2)dppz-idzo](2+), complex 1) in buffer solution. Polyanionic heparin is found to interact with a positively charged Ru-complex through electrostatic effects and/or hydrogen bonding interactions, which leads to a significant fluorescence enhancement of the Ru-complex. To get insight into this fluorescence "switch on" behavior, the binding model of the Ru-complex to heparin is established by employing molecular docking simulations based on the fluorescence and UV absorption results. The selectivity results of the fluorescence assay reveal that our complex displayed good fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya), which have lower charge density and/or structural compatibility as compared to that of heparin. Quantification of heparin is also performed and a linear calibration curve is observed in the range of 0.01-4.87 U mL(-1) (the limit of detection is 0.01 U mL(-1)) for heparin detection in diluted FBS solution. This "one-step" fluorescence "switch on" assay for heparin detection is label-free, convenient, sensitive and selective, and has a long emission wavelength and large Stokes shift.

Investigation on the aggregation behaviors and filament morphology of tau protein by a simple 90° angle light-scattering assay.

The in vitro aggregation of tau constructs was monitored by a simple 90° angle light-scattering (LS) approach which was conducted directly on fluorescence instrument. At the optimum incident wavelength (550 nm, unpolarized), the sensitivity of LS was high enough to detect tau aggregation at micromolar range. The nucleation and elongation, different events in the aggregation process of 4RMBD construct (corresponding with the four repeated units of tau Microtubule Binding Domain) could be observed by this approach, as compared with ThS fluorescence assay. The validity of this technique was demonstrated over a range of tau concentrations with different tau filaments. Linear regression of scattering light against concentration yielded the x-intercept, the critical concentrations of tau constructs. The critical concentrations of 4RMBD and its S305N mutant are 5.26 µM and 4.04 µM respectively, indicating point mutation S305N, which is associated with FTDP-17, appear to enhance the heparin-induced tau aggregation in vitro. Furthermore, the slopes of concentration dependence curves, as well as the angle dependence, were discussed based on the filaments morphology examined by electron microscopy and ultrasonication experiment.

A naked-eye on-off-on molecular "light switch" based on a reversible "conformational switch" of G-quadruplex DNA.

Herein, we report a new strategy for developing an on-off-on molecular "light switch" by utilizing the pH value to control the "conformational switch" of G-quadruplex DNA. A novel ruthenium(II) complex with an emission enhancement factor of 150 was synthesized and introduced to detect the switch by the naked eye. The "light switch" can be repeatedly cycled off and on through the addition of H(+) and OH(-), respectively. The conformational transitions of G-quadruplex DNA in K(+) solution at different pH values in the acidic region were evidenced by circular dichroism and fluorescence titrations. Computational calculations by applying density functional theory (DFT)/time-dependent DFT and molecular docking were also carried out to gain insight into the "light-switch" mechanism.

Cooperative folding of tau peptide by coordination of group IIB metal cations during heparin-induced aggregation.

The group IIB elements, especially Cd(II) and Hg(II), are increasingly considered as potential environmental neurotoxins. This study demonstrates that the Alzheimer's tau fragment R2, corresponding to the second repeat of the microtubule-binding domain, can bind to Zn(II), Cd(II) and Hg(II). Isothermal titration calorimetry experiments suggest that the most likely coordination site is the thiol group of Cys291, and this is further confirmed by a control experiment using a C291A mutant peptide. Circular dichroism spectrum reveals that the coordination of group IIB cations, especially Hg(II), can induce pronounced conformational conversions in natively unfolded R2, from random coil to other ordered structures. ThS fluorescence assays and electron microscopy indicate that the group IIB cations promote heparin-induced aggregation of R2, giving relatively small R2 filaments. The efficiency in promoting aggregation, as well as inducing conformational conversion, varies strongly with the cation's polarizability. Based on these results, a model is proposed in which the cooperative folding of R2 through cross-bridging of group IIB cations is suggested to be a key factor in promoting aggregation, in addition to the effective neutralization of coulombic charge-charge repulsion by heparin, the poly-anion inducer. Our results provide clues to understanding the potential pathogenic role of group IIB metals in the development of neurofibrillary tangles, a typical hallmark of Alzheimer's disease.

[The effects of Guanxinshutong on protection of left ventricular function after acute myocardial infarction in rats].

OBJECTIVE: To assess the effects of Guanxinshutong capsule (GXST) on protection of left ventricular (LV) function after acute myocardial infarction (AMI) in rats. METHODS: Twenty-eight male Sprague Dawley rats were randomized to Model group, Drug group and Sham-operated group, with acute myocardial infarction (AMI) achieved by ligating coronary artery in Model and Drug groups. From one week before surgery to four weeks after surgery, GXST for Drug group (1.5 g/kg, 2 times/day) or saline for Model and Sham-operated groups was administered via direct gastric gavage. After four weeks of treatment following surgery, measurement of LV function, pathohistological observation and analysis were performed. RESULTS: Compared with rats in the Model group, LV systolic pressure (LVSP) [(97.7 ± 9.0) mm Hg (1 mm Hg = 0.133 kPa) vs (85.9 ± 9.4) mm Hg], the maximum rising rate of LV pressure (+dp/dtmax) [(4810.2 ± 595.0) mm Hg/s vs (3786.2 ± 723.0) mm Hg/s] and the maximum dropping rate of LV pressure (-dp/dtmax) [(3781.6 ± 573.6) mm Hg/s vs (2774.4 ± 633.5)mm Hg/s] in the Drug group were significantly increased, while LV end-diastolic pressure (LVEDP) [(10.3 ± 0.7) mm Hg vs (12.7 ± 2.4) mm Hg] in the Drug group was significantly decreased (all P < 0.05). Myocardial pathohistological morphology was improved in the Drug group with fibrosis alleviated [(5.13 ± 1.37)% vs (7.27 ± 1.01)%] and infarct size reduced [(20.14 ± 8.49)% vs (31.90 ± 4.98)%]. Apoptosis index (AI) was decreased [(14.05 ± 4.04)% vs (20.87 ± 6.03)%] and vessel density was significantly increased by 1.48-fold in the Drug group (all P < 0.05). CONCLUSIONS: GXST is effective in protecting LV function after AMI in rats, which may be affect through increasing vessel density of infarction area, improving myocardial pathohistological morphology, alleviating fibrosis, reducing infarct size and decreasing AI.

Synthesis, characterization, and DNA-binding of chiral complexes delta- and lambda-[Ru(bpy)2(pyip)]2+.

New chiral Ru(II) complexes delta and lambda-[Ru(bpy)(2)(pyip)](PF(6))(2) [(bpy = 2,2'-bipyridine; pyip = (2-(1-pyrenyl)-1H-imidazo[4,5-f] [1,10]phenanthroline] were synthesized and characterized by elemental analysis, (1)H NMR, ESI-MS, IR, and CD spectra. Their DNA-binding properties were studied by means of UV-vis, emission spectra, CD spectra and viscosity measurements. A subtle but detectable difference was observed in the interaction of both enantiomer with CT-DNA. Spectroscopy experiments indicated that each of these complexes could interact with the DNA. The DNA-binding of the Delta-enantiomer was stronger than that of Lambda-enantiomer. DNA-viscosity experiments provided evidence that both Delta- and Lambda-[Ru(bpy)(2)(pyip)](PF(6))(2) bound to DNA by intercalation. At the same time, the DNA-photocleavage properties of the complexes were investigated too. Under irradiation with UV light, Ru(II) complexes showed different efficiency of cleaving DNA.

Luminescent Ru(ii)-thiol modified silver nanoparticles for lysosome targeted theranostics.

Silver nanoparticles (AgNPs) modified by luminescent Ru(ii) complexes not only possess bright red fluorescence but also can target lysosomes. Cell imaging and a cytotoxicity study suggest that Ru1-2·AgNPs may act as a potential theranostic agent.

G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands.

In this paper, three new Ruthenium(II) polypyridyl complexes containing ascididemin (ASC) as main ligand have been synthesized and characterized. Their interactions with different G-quadruplex (Htelo, c-myc and c-kit) (Htelo: human telomeric DNA, c-myc: cellular-myelocytomatosis viral oncogene, c-kit: oncogene c-kit promoter sequences) and duplex (ds26) DNA sequences were comparatively studied with the free ligand ASC by a series of spectroscopic techniques including UV-vis (ultraviolet-visible) spectroscopy, FID (fluorescent intercalator displacement) assay, and FRET (fluorescence resonance energy transfer) melting assay. Molecular docking studies were also performed to support the binding mode of the compounds with G-quadruplex DNA. Results indicated that [Ru(bpy)2ASC]·(PF6)2 (1), [Ru(phen)2ASC]·(PF6)2 (2), [Ru(tatp)2ASC]·(PF6)2 (3) (bpy = 2,2'­bipyridine, phen = 1,10­phenanthroline, tatp = 1,4,8,9­tetra­aza­triphenylene) and ASC can effectively bind G-quadruplex and duplex DNA and stabilization ability lies in the order 3 > 2 > 1 > ASC. Complex 3 was determined to be the most promising candidate for further in vitro studies and potential anticancer drug.

Impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau fragment corresponding to the third repeat of microtubule-binding domain.

Environmental exposure to some heavy metals such as cadmium appears to be a risk factor for Alzheimer's disease (AD), however, definite mechanism of their toxicity in AD remains to be elucidated. Previous studies largely focused on the metal ions binding to beta-amyloid, however, very few papers concerned the interaction between tau and metal ions. For the first time, we investigated the impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau peptide R3, corresponding to the third repeat of microtubule-binding domain. The initial state of R3 was proven to be dimeric linked by intermolecular disulfide bond, in the non-reducing buffer (Tris-HCl buffer pH7.5, containing no reducing reagent). In this paper, we show that Cd(II) can accelerate heparin-induced aggregation of R3 or independently induce the aggregation of R3, as monitored by ThS fluorescence. In the presence of Cd(II), the resulting R3 filaments became much smaller, as revealed by electron microscopy. Binding to the Cd(II) ion, the dimeric R3 partially lost its random coil, and converted to alpha-helix structure, as revealed by CD and Raman spectrum. Stoichiometric analysis of CD signal against the ratio of [Cd(II)]/[R3] suggested that the coordination intermediate consisted of two R3 dimers binding to a central cadmium ion. As the seed, the coordination intermediate could extensively accelerate the self-aggregation of R3 via promoting the nucleation step. On the other hand, gain in alpha-helix structure on the peptide chain, by coordinating with Cd(II), could be a critical role to promote self-aggregation, as revealed by Raman spectrum. These results provide a further insight into the mechanism of tau filament formation and emphasize the possible involvement of Cd(II) in the pathogenesis of AD.

1,2-Bis[amino-(pyrimidin-2-yl)methyl-ene]hydrazine dihydrate.

The centrosymmetric organic molecule in the title compound, C(10)H(10)N(8)·2H(2)O, is essentially flat and has a trans configuration. The mol-ecules are linked by inter-molecular O-H⋯N, N-H⋯O and N-H⋯N hydrogen bonds to form a linear chain structure.

Integration of G-quadruplex and DNA-templated Ag NCs for nonarithmetic information processing.

To create sophisticated molecular logic circuits from scratch, you may not believe how common the building blocks can be and how diverse and powerful such circuits can be when scaled up. Using the two simple building blocks of G-quadruplex and silver nanoclusters (Ag NCs), we experimentally construct a series of multifunctional, label-free, and multi-output logic circuits to perform nonarithmetic functions: a 1-to-2 decoder, a 4-to-2 encoder, an 8-to-3 encoder, dual transfer gates, a 2 : 1 multiplexer, and a 1 : 2 demultiplexer. Moreover, a parity checker which is capable of identifying odd and even numbers from natural numbers is constructed conceptually. Finally, a multi-valued logic gate (ternary inhibit gate) is readily achieved by taking this DNA/Ag NC system as a universal platform. All of the above logic circuits share the same building blocks, indicating the great prospects of the assembly of nanomaterials and DNA for biochemical logic devices. Considering its biocompatibility, the novel prototypes developed here may have potential applications in the fields of biological computers and medical diagnosis and serve as a promising proof of principle in the not-too-distant future.

Coordination polymer nanoparticles from nucleotide and lanthanide ions as a versatile platform for color-tunable luminescence and integrating Boolean logic operations.

Novel supramolecular coordination polymer nanoparticles (CPNs) were synthesized via the self-assembly of guanosine monophosphate (GMP) and lanthanide ions (Ln3+, including Tb3+, Eu3+ and Ce3+) in aqueous solution. These CPNs (GMP/Tb3+, GMP/Eu3+ and GMP/Ce3+) have an identical coordination environment but exhibit completely different luminescence properties responding to external stimuli such as dipicolinic acid (DPA), ethylene diamine tetraacetic acid (EDTA), pH and metal ions, which has inspired us to tune the emission color of the CPNs and perform multiple logic operations. Firstly, color-tunable luminescence from red to green can be easily achieved by modulating the doping ratio of Tb3+ and Eu3+ into GMP. Notably, trichromatic white light emitting CPNs can be successfully realized by simultaneously doping Tb3+, Eu3+ and Ce3+ into the host or just adjusting the pH of the solution. What's more, by employing GMP/Tb3+ CPNs as a logic operator, we have achieved the implementation of multilayered gate cascades (INH-INH, NOR-OR). When GMP/Eu3+ CPNs served as a logic operator, the logic elements can be integrated as another combinatorial gate (AND-INH). Moreover, by employing the red emission of Eu3+ and blue emission of GMP as the dual-output signal transducer, a set of parallel logic gates was established successfully. These results help elucidate the design rules by which simple logic can be integrated to construct cascaded logic gates and expand the applications of CPNs in light-emitting diode (LED) lamps and biological systems.

A RET-supported logic gate combinatorial library to enable modeling and implementation of intelligent logic functions.

Boolean logic gates integrate multiple digital inputs into a digital output. Among these, logic gates based on nucleic acids have attracted a great deal of attention due to the prospect of controlling living systems in the way we control electronic computers. Herein, by employing Thioflavin T (ThT) as a signal transducer, we integrated multiple components based on RET (a type of proto-oncogene) into a logic gate combinatorial library, including basic logic gates (NOR, INHIBIT, IMPLICATION), a single three-input NOR gate, and combinatorial gates (INHIBIT-OR, NOT-AND-NOR). In this library, gates were connected in series where the output of the previous gate was the input for the next gate. Subsequently, by taking advantage of the library, some intelligent logic functions were realized. Expectedly, a biocomputing keypad-lock security system was designed by sequential logic operations. Moreover, a parity checker which can identify even numbers and odd numbers from natural numbers was established successfully. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between different gates. Together, our system may serve as a promising proof of principle that demonstrates increased computational complexity by linking multiple logic gates together.

Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system.

We report here an ultrasensitive strategy based on the recognition-induced conformational alteration of aptamer and fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequence in proximity to silver nanoclusters for adenosine triphosphate (ATP), adenosine (A) and thrombin (TB) detection. Herein, we designed two tailored DNA sequences noted as complementary DNA (abbreviated as c-DNA) and signal probe DNA (abbreviated as s-DNA), respectively. c-DNA is designed as a special structure consisting of a sequence complementary to aptamer at the 3'-end and a guanine-rich DNA sequence at the 5'-end; s-DNA contains a cytosine-rich sequence responsible for Ag NCs templated synthesis at the 3'-end and a link sequence (part of aptamer) complementary to partial of the c-DNA at the 5'-end. In the presence of target, the aptamer associated with the target, resulting in the formation of duplex DNA (dsDNA), the DNA-Ag NCs thereafter could close to the guanine-rich sequence, leading to enhanced fluorescence signal readout. The widespread application of the sensing system is achieved success in the detection of three biomolecules. ATP, adenosine and thrombin in the range of 0.5-8.0 µM, 0.5-7.0 µM and 50-900 nM could be linearly detected with the detection limits of 91.6 nM, 103.4 nM and 8.4 nM, respectively. This label-free and turn-on fluorescent sensing system employing the mechanism proposed here turns out to be sensitive, selective, and convenient for the detection of biomolecules without washing and separation steps.

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation.

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.

Impacts of terminal modification of [Ru(phen)2dppz](2+) on the luminescence properties: a theoretical study.

[Ru(phen)2dppz](2+) and other closely related ruthenium(II) complexes containing π-extended ligands were found to be non or weakly emissive in water, while exhibiting significant luminescence intensity growth when bound to DNA, however, a satisfactory interpretation has not been provided on this "light switch" mechanism. In the present study, we investigated the vertical transitions and triplet excited states of [Ru(phen)2dppz](2+) (1), [Ru(phen)2dppzi](2+) (2) and [Ru(phen)2dppz-idzo](2+) (3) in the gas phase and aqueous solution, through time dependent-density functional theory (TDDFT). Based on the optimized (3)MLCT and (3)LLCT structures and energies, we found that the (3)MLCT state might be responsible for the emissions of the complexes. Interesting connections between the singlet vertical transitions and the luminescence properties were noticed. Through ZORA-TDDFT calculation with perturbative SOC, we evaluated the intersystem crossing between the lowest singlet excited state, and both (3)MLCT state and (3)LLCT state, which gave a reasonable explanation for the luminescence properties of these complexes.

Alginate-calcium microsphere loaded with thrombin: a new composite biomaterial for hemostatic embolization.

To date, transcatheter arterial embolization (TAE) has become a standard treatment to control intracavitary bleeding as an alternative to surgery. Due to excellent biocompatibility and no residual in vivo, biodegradable materials are preferred in TAE. However, gelfoam is the only commercially available biodegradable embolic material used to treat blunt trauma of solid abdominal viscera until now, and controversial on its stability and reliability never stopped in the past five decades. In this study, a new biodegradable macromolecule material (thrombin-loaded alginate-calcium microspheres, TACMs) was prepared using electrostatic droplet techniques and a special method was developed for hemostatic embolization. Thrombin was successfully loaded into microspheres with high encapsulation efficiency and drug loading capacity. A burst release of TACMs was observed at early stage and sustained release later on, with the activity of thrombin preserved well. The strength of TACMs mixed thrombus, which was used as embolic agent, increased in a dose-dependent manner after TACMs were added. In addition, the TACMs were verified to be of no cytotoxicity and systemic toxicity, and biodegradable in vivo. Finally, the results of preliminary applications revealed that the TACMs could serve as an effective and promising embolic material for blunt trauma and hemorrhage of solid abdominal viscera.

Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods.

The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the four-repeat microtubule binding domain (4RMBD) in tau protein and its three mutants, 4RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4RMBD mutation.