Impact of lyoprotectors on protein-protein separation in the solid state: Neutron- and X-ray-scattering investigation

BackgroundPolyhydroxycompounds (PHC) are used as lyoprotectors to minimize aggregation of pharmaceutical proteins during freeze-drying and storage.MethodsLysozyme/PHC mixtures with 1:1 and 1:3 (w/w) ratios are freeze-dried from either H₂O or D₂O solutions. Disaccharides (sucrose and trehalose), monosaccharide (glucose), and sugar alcohol (sorbitol) are used in the study. Small-angle neutron and X-ray scattering (SANS and SAXS) are applied to study protein-protein interaction in the freeze-dried samples.ResultsProtein interaction peak in the freeze-dried mixtures has been detected by both SANS (D₂O-based samples only) and SAXS (both D₂O- and H₂O-based). In the 1:1 mixtures, protein separation distances are similar (center-of-mass distance of approx. 31 Å) between all lyoprotectors studied. Mixtures with a higher content of the disaccharides (1:3 ratio) have a higher separation distance of approx 40 Å. The higher separation could reduce protein-protein contacts and therefore be associated with less favourable aggregation conditions. In the 1:3 mixtures with glucose and sorbitol, complex SANS and SAXS/WAXS patterns are observed. The pattern for the glucose sample indicate two populations of lysozyme molecules, while the origin of multiple SAXS peaks in the lysozyme/sorbitol 1:3 mixture is uncertain.ConclusionsProtein-protein separation distance is determined predominantly by the lyoprotector/protein weight ratio.General significanceUse of SANS and SAXS improves understanding of mechanisms of protein stabilization by sugars in freeze-dried formulations, and provide a tool to verify hypothesis on relationship between protein/protein separation and aggregation propensity in the dried state.     

Tadalafil Integrates Nitric Oxide-Hydrogen Sulfide Signaling to Inhibit High Glucose-induced Matrix Protein Synthesis in Podocytes

Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H₂S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H₂S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase β. Tadalafil rapidly increased the expression and activity of the H₂S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H₂S and nitric oxide (NO). N^ω-Nitro-l-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H₂S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H₂S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.     

Palladium(II) and platinum(II) complexes of 2-hydroxy acetophenone N(4)-ethylthiosemicarbazone - crystal structure and description of bonding properties

Reaction of H₂PtCl₄ and K₂PdCl₄ with 2-hydroxyacetophenone N(4)-ethylthiosemicarbazone, H₂Ap4Et, afforded [Pt(Ap4Et)(H₂Ap4Et)] and [Pd(Ap4Et)(H₂Ap4Et)]. Their crystal and molecular structures are reported and represent the first 1:2 thiosemicarbazone complexes with ligands having both different formal charge and denticity. The dianion, Ap4Et²⁻, coordinates in a planar conformation to palladium(II) or platinum(II) via the phenolato O, imine N and thiolato S atoms, while the neutral molecule exhibits monodentate coordination by the thione S atom. Intra-, intermolecular hydrogen bonds and C-H?π contacts lead to aggregation and a supramolecular assembly. Electronic, IR, and NMR spectral data, as well as electrochemical measurements, are included. The pK_a values of the poorly water soluble H₂Ap4Et were obtained spectrophotometrically in aqueous solutions of constant ionic strength.     

Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His₆-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2_SH3) and a mutant that adopts an intrinsically ‘open’ and activated conformation (CB2_{SH3/W24A-E262A}), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.     

Cytotoxicity studies of [PtCl2(H2bim)] (H2bim = 2,2′-biimidazole): Study of its interaction with a small protein PCI (potato carboxypeptidase inhibitor)

The effect of the platinum compound [PtCl₂(H₂bim)] (H₂bim = 2,2′-biimidazole) on the plasmid DNA conformation was previously studied by electrophoresis in agarose gel and on calf thymus DNA by circular dichroism spectroscopy. The effect of this compound on pBR322 plasmid DNA has now been visualized by atomic force microscopy, which shows that the complex modifies the DNA in the same way as cisplatin does. The cytotoxic activity of [PtCl₂(H₂bim)] in HeLa-229, HL-60, A2780 and A2780cisR cell lines has also been evaluated. Likewise, the interaction of [PtCl₂(H₂bim)] with the small protein potato carboxypeptidase inhibitor (PCI) and a PCI mutant in which glycine 39 was substituted by methionine has been followed by HPLC/mass spectrometry. The interaction with the mutant protein PCI showed the formation of monofunctional adducts that ultimately gave bifunctional adducts. PCI mutant protein could be a good carrier of this platinum compound to the tumour cells in which the antiproliferative behaviour was demonstrated.     

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics: INVESTIGATION AT SINGLE MOLECULAR RESOLUTION*

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r_H) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r_H of 29 Å) over an extended one (r_H of 40 Å), which forms in their absence. Also, the time constant of a kinetic component (τ_R) of about 30 μs has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over and behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.     

Hydrogen sulfide concentration in the milieu of the hydrated alanine dipeptide determines its polyproline II-beta propensity: Main chain contribution to the energetic origin of the formation of amyloid

In view of the observation that the concentration of hydrogen sulfide in brains with Alzheimer’s disease (AD) is lower than that in normal brains and in line with our previous studies indicating that additional content in the aqueous environment (milieu) of a peptide can change its local energetic preference from a polyproline II (P) to a β conformation (and therefore its tendency to form the β-chain structures that lead to the amyloid plaques associated with the disease), we have studied the effect of H₂S concentration on such propensity in a simple model peptide, the alanine dipeptide (ADP). The two concentration states are represented by ADP(H₂O)₁₈(H₂S) and ADP(H₂O)₁₈(H₂S)₂. Ab initio calculations of these structures show that the lowest energy of the former is a β conformation while that of the latter is a P, mirroring the observed AD results and strengthening our proposal that amyloid diseases are better viewed in the context of a protein milieu-folding paradigm.     

Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

Sulfide Oxidation by a Noncanonical Pathway in Red Blood Cells Generates Thiosulfate and Polysulfides

A cardioprotectant at low concentrations, H₂S is a toxin at high concentrations and inhibits cytochrome c oxidase. A conundrum in H₂S homeostasis is its fate in red blood cells (RBCs), which produce H₂S but lack the canonical mitochondrial sulfide oxidation pathway for its clearance. The sheer abundance of RBCs in circulation enhances the metabolic significance of their clearance strategy for H₂S, necessary to avoid systemic toxicity. In this study, we demonstrate that H₂S generation by RBCs is catalyzed by mercaptopyruvate sulfurtransferase. Furthermore, we have discovered the locus of sulfide oxidation in RBCs and describe a new role for an old protein, hemoglobin, which in the ferric or methemoglobin state binds H₂S and oxidizes it to a mixture of thiosulfate and hydropolysulfides. Our study reveals a previously undescribed route for the biogenesis of hydropolysulfides, which are increasingly considered important for H₂S-based signaling, but their origin in mammalian cells is unknown. An NADPH/flavoprotein oxidoreductase system restores polysulfide-carrying hemoglobin derivatives to ferrous hemoglobin, thus completing the methemoglobin-dependent sulfide oxidation cycle. Methemoglobin-dependent sulfide oxidation in mammals is complex and has similarities to chemistry reported for the dissolution of iron oxides in sulfidic waters and during bioleaching of metal sulfides. The catalytic oxidation of H₂S by hemoglobin explains how RBCs maintain low steady-state H₂S levels in circulation, and suggests that additional hemeproteins might be involved in sulfide homeostasis in other tissues.     

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn²⁺-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.     

Hydrogen sulfide and rhizobia synergistically regulate nitrogen (N) assimilation and remobilization during N deficiency-induced senescence in soybean

Hydrogen sulfide (H₂S) is emerging as an important signalling molecule that regulates plant growth and abiotic stress responses. However, the roles of H₂S in symbiotic nitrogen (N) assimilation and remobilization have not been characterized. Therefore, we examined how H₂S influences the soybean (Glycine max)/rhizobia interaction in terms of symbiotic N fixation and mobilization during N deficiency-induced senescence. H₂S enhanced biomass accumulation and delayed leaf senescence through effects on nodule numbers, leaf chlorophyll contents, leaf N resorption efficiency, and the N contents in different tissues. Moreover, grain numbers and yield were regulated by H₂S and rhizobia, together with N accumulation in the organs, and N use efficiency. The synergistic effects of H₂S and rhizobia were also demonstrated by effects on the enzyme activities, protein abundances, and gene expressions associated with N metabolism, and senescence-associated genes (SAGs) expression in soybeans grown under conditions of N deficiency. Taken together, these results show that H₂S and rhizobia accelerate N assimilation and remobilization by regulation of the expression of SAGs during N deficiency-induced senescence. Thus, H₂S enhances the vegetative and reproductive growth of soybean, presumably through interactions with rhizobia under conditions of N deficiency.     

Synthesis, crystal structure, and catalytic studies on dinuclear copper(II) mesocates

Imine based bis-bidentate ligands H₂-m-xysal, (L₁H₂); H₂-m-xysal-Cl, (L₂H₂); H₂-m-xysal-Br, (L₃H₂); H₂-m-xysal-OCH_3, (L₄H₂); H₂-m-xysal-(t-Bu)_2, (L₅H₂) were synthesized and characterized. These substituted 1,3-bis(hydroxylbenzyl)-diaminoxylene dianion ligands upon treating with copper(II) acetate in 2:2 equivalent of L:M ratio, resulted in a series of binuclear [Cu₂(m-xysal)₂] neutral complexes 1-5. The crystal structures determined for the complexes 1 and 2 indicate a dinuclear association. The CH?π interaction observed between the metal-chelate ring and the hydrogens associated with m-xylene spacer moiety being first in this series of complexes, is demonstrated to stabilize the helical conformation through intramolecular self assembly process. The position of the resonance on the EPR spectra and the absence of ΔMs = ±1 feature for the complexes 2, 3, and 5 obtained for room temperature solid state samples revealed that the metal centers though exist in the dinuclear unit, they are separated from each other and possess a non-interacting monomer-type metal-metal association. The Cu(II) centers in all these complexes possessing an intermediate geometry between tetrahedral and square planar, an appropriate catalytic study converting 4-nitrobenzaldehye to corresponding nitroaldol was carried out using complex 5.     

Formation and Decay of the Arrestin·Rhodopsin Complex in Native Disc Membranes

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (G_t). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of G_t to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to G_t-bound Meta II. In arrestin, a modest loss of β-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.     

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg²⁺ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from Tb_bVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within Tb_bVSP1.     

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H₂O₂-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H₂O₂-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H₂O₂-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.     

T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.     

Endogenous CBS–H<Subscript>2</Subscript>S Pathway Contributes to the Development of CCI-Induced Neuropathic Pain

Studies showed a complex relationship between hydrogen sulfide (H₂S) and neuropathic pain. In this study, the relationship between endogenous CBS–H₂S pathway in L_4–6 spinal cord and neuropathic pain was explored. A total of 163 adult Kunming mice were used in this study. CBS expression and H₂S formation in L_4–6 spinal cord were detected in the development of neuropathic pain firstly. Then, effect of AOAA, an CBS inhibitor, on treatment of neuropathic pain by chronic construction injury surgery (CCI) was detected. Pain thresholds and activation of NF-κB(p65), ERK1/2 and CREB were measured as biomarks of neuropathic pain. Results showed that CCI surgery significantly upregulated protein expression of CBS and H₂S formation. Correlation analysis showed pain thresholds had negative relationships with protein expression of CBS and H₂S formation. Treatment with AOAA, a CBS inhibitor, inhibited CCI-induced upregulation of CBS expression and H₂S formation (P < 0.05). Further, AOAA significantly decreased activation of NF-κB(p65), ERK1/2 and CREB pathway, and reversed CCI-induced allodynia (P < 0.05). This indicated that CBS–H₂S pathway promoted the development of neuropathic pain. CBS–H₂S pathway could be a promising target for treatment of neuropathic pain.     

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA^val, and yeast tRNA^phe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.     

Interaction between lanthanum ion and horseradish peroxidase in vitro

The interaction between lanthanum ion (La³⁺) and horseradish peroxidase (HRP) in vitro was investigated using a combination of biophysical and biochemical methods. When the molar ratio of La³⁺ and HRP is low, it was found that the interaction between La³⁺ and HRP mainly depends on the electrostatic attraction, van der waals force and hydrogen bond etc. Thus, the interaction is weak and the La–HRP complex cannot be formed in vitro. As expected, the interaction can change the conformation of HRP molecule, leading to the increase in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Therefore, the catalytic activity of HRP for the H₂O₂ reduction is improved. When the molar ratio of La³⁺ and HRP is high, La³⁺ can strongly coordinate with O and/or N in the amide group of the polypeptide chain of HRP molecule, forming the La–HRP complex. The formation of the La–HRP complex causes the change in the conformation of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the catalytic activity of HRP for the H₂O₂ reduction is decreased comparing with that of HRP in the absence of La³⁺. The results can provide some references for understanding the interaction mechanism between trace elements ions and peroxidase in living organisms.     

Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry

The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (α-Fe₂O₃). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 × 90 × 65 ų. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ∼7 Å (the maximum dimension was 96 Å for oxidized OmcA, and 89 Å for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes.