High Α-helical Content Research Articles

Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn2+ ions

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn2+. By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket.

Structural Insights into the Dimeric Form of Bacillus subtilis RNase Y Using NMR and AlphaFold.

RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics and biophysical analysis have previously shown that Nter-BsRNaseY, which is in equilibrium between a monomeric and a dimeric form, displays an elongated fold with a high content of α-helices. Using multidimensional heteronuclear NMR and AlphaFold models, here, we show that the Nter-BsRNaseY dimer is constituted of a long N-terminal parallel coiled-coil structure, linked by a turn to a C-terminal region composed of helices that display either a straight or bent conformation. The structural organization of the N-terminal domain is maintained within the AlphaFold model of the full-length RNase Y, with the turn allowing flexibility between the N- and C-terminal domains. The catalytic domain is globular, with two helices linking the KH and HD modules, followed by the C-terminal region. This latter region, with no function assigned up to now, is most likely involved in the dimerization of B. subtilis RNase Y together with the N-terminal coiled-coil structure.

Single p197 molecules of the mitochondrial genome segregation system of Trypanosoma brucei determine the distance between basal body and outer membrane

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the ∼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana

BackgroundCP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background.MethodsA combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12.ResultsHere, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions.ConclusionsThese findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle.Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta.While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism.5h3nzM1M4bjv4RCQxTYJACVideo

MON-175 Structural Instability as an Underlying Pathomechanism in Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders affecting key enzymes of cortisol biosynthesis. In the majority of cases the underlying cause are detrimental mutations in the steroidogenic cytochrome P450 enzyme 21-hydroxylase (CYP21A2). Early diagnosis via newborn screening programs in most Western countries and lifelong oral cortisol replacement therapy enable survival, however quality of life often is reduced and co-morbidities are substantially increased. Treatment is a major challenge as disease control can only be achieved with supraphysiological glucocorticoid doses. In addition, the currently available drugs cannot ideally mimic the circadian rhythm and stress adaption of cortisol secretion. Currently, disease severity is classified by residual enzyme activity. The goal of our research is to better understand the specific biophysico-chemical pathomechanism of 21-hydroxylase deficiency in order to enable causative therapeutic approaches. To this end, we investigated the structural and stability properties of six clinically relevant mutant variants of CYP21A2 (V282G/L, P31L, D323G, R484Q/W). Difficulty in purification of these CYP21A2 variants and various biophysical studies suggest that the proteins were less stable than wild-type (WT). Structural and thermal stability assessment by circular dichroism (CD) spectroscopy of recombinant, purified CYP21A2 mutant variants revealed high α-helical content for the WT (65% α-helix) and the mutants at the position 282 (V282G: 60.6 %, V282L: 57.6%). Other mutations (P31L, D323G, R484Q/W) disrupt the α-helical organization of CYP21A2 in exchange for a slight increase in ß-sheet content but mainly for random coil. Temperature dependent CD spectroscopy showed that all mutant variants have reduced thermal stability (Tm: 41.3 - 45,6°C) compared to the WT (Tm: 47.1°C). Tryptophane fluorescence showed that mutant variants of the protein were more prone to local unfolding at the hydrophobic core compared to WT using urea as denaturant. Furthermore, in UV/Vis spectroscopy at 280 nm and 418 nm we could demonstrate that all mutant variants had a reduced heme incorporation (A418/A280: 0.20 - 0.63) compared to WT (A418/A280: 0.88). Our results show that correct structural folding and stability pose a major problem in specific mutations involved in CAH. Therefore we propose that structural protein instability, play a key role in the pathophysiology of CAH and thus might constitute a novel tailored therapeutic target for the treatment of affected patients.

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.

Computational study of solution behavior of magainin 2 monomers

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.

Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target

100K is an important scaffolding protein of adenoviruses including fowl adenovirus serotype 4 (FAdV-4) that causes inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in poultry. 100K carries out the trimerization of the major capsid hexon protein of the virus for the generation of new virions inside the target host cells. Despite its critical role for FAdV-4, no structural study, in particular, has been conducted so far. Here, the overexpression of soluble 100K protein was successfully carried out in E. coli using various expression constructs and purification yield of 3mg per litre culture volume was obtained. Gel filtration chromatography suggested that 100K protein exists in trimeric form. Circular dichroism and Fourier transform infrared spectroscopy clearly reveal that 100K protein folds with a high content of α-helices. The 3-dimentional homology model of the 100K protein, refined with molecular dynamics tools also depicts higher α-helical content within the protein model. Moreover, overexpressed recombinant 100K protein could be used to differentiate vaccinated and FAdV-4 infected chickens on the basis of higher serum anti 100K antibody titres. Our work provides preliminary structural and functional results to study biological role of the 100K protein and for further investigations to develop 100K inhibitors to control IBH-HPS in poultry.

Purification and Characterization of 2S Albumin from Seeds of Wrightia tinctoria Exhibiting Antibacterial and DNase Activity.

2S albumin is a low-molecular-weight seed storage protein belonging to the prolamin superfamily. In the present work a small 2S albumin (WTA) protein of ~16 kDa has been purified from the seeds of Wrightia tinctoria. The WTA is a heterodimer protein with a small subunit of ~5 kDa and a larger subunit of ~11 kDa bridged together through disulphide bonds. The protein exhibits deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The protein also showed antibacterial activity against Morexalla catarrhalis. CD studies indicate a high α-helical content in the protein. The conserved disulphide bonds in the protein suggest that the WTA is highly stable under high pH and temperature like other 2S albumin.

Secondary Structure Preferences of the Anthrax Toxin Protective Antigen Translocase

In order for many proteins to move across hydrophobic membrane bilayers, they must be unfolded and translocated by a membrane-embedded channel. These translocase channels interact with the substrate proteins they translocate via hydrophobic pore loops and cleft structures called clamps. The molecular basis for how clamps facilitate unfolding and translocation is poorly understood. Anthrax toxin is composed of three proteins, a translocase channel-forming subunit, called protective antigen (PA), and two substrate proteins, called lethal factor (LF) and edema factor. Oligomeric PA forms a large channel that contains three types of polypeptide clamp sites: an α clamp, a phenylalanine clamp, and a charge clamp. Currently, it is thought that these clamp sites operate allosterically and promote translocation via an allosteric helix compression mechanism. Here, we report on the substrate secondary structure dependence of the PA channel. Peptides derived from regions of LF with high α-helical content bound cooperatively, but those derived from β-sheet regions in LF did not, suggesting that an allosteric site preferentially recognizes α-helical structure over β-sheet structure. Peptides derived from helical sites in LF showed increasingly longer single-channel blockades as a function of peptide concentration, a result that was consistent with stronger clamping behavior and reduced backsliding. Moreover, peptides derived from helical regions of LF translocated more efficiently than peptides derived from β-sheet regions of LF. Overall, in support of the allosteric helix compression model, we find that the channel prefers α-helical sequences over β-sheet sequences.

Chemical and thermal influence of the [4Fe–4S]2 + cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis

The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe–4S]2+ cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe–4S]2+ cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe–4S]2+ cluster. UV–vis and fluorescence spectroscopy results demonstrate that between pH3.0 and 4.0 the integrity of the [4Fe–4S]2+ cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45°C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Biophysical characterization of polyomavirus minor capsid proteins

The murine polyomavirus encodes three structural proteins, VP1, VP2 and VP3, which together form the viral capsid. The outer shell of this capsid is composed of the major capsid protein VP1, the inner shell consists of VP2 and its N-terminally truncated form VP3. These two minor capsid proteins interact with their identical C-terminal part in the central cavity of VP1 pentamers, building the capsid assembly unit. While the VP1 structure and functions are well studied, VP2 and VP3 are poorly understood. In order to get a detailed insight into the structure and function of the minor capsid proteins, they were produced recombinantly in Escherichia coli as inclusion bodies and refolded in vitro. The success of refolding was strictly dependent on the presence of detergent in the refolding buffer. VP2 and VP3 are monomeric and their structures exhibit a high α-helical content. The function of both proteins could be monitored by complex formation with VP1. Furthermore, we could demonstrate a hemolytic activity of VP2/VP3 in vitro, which fits well into a postulated membrane interaction of VP2 during viral infection.

High-level coproduction, purification and characterisation of laccase and exopolysaccharides by Coriolus versicolor

In this study, a two-stage pH-shift fermentation process was developed for the coproduction of laccase and exopolysaccharides (EPS) by Coriolus versicolor. At the same time, laccase and EPS were purified and characterised in detail. The results showed that the highest laccase and EPS production reached 7680Ul−1 and 8.2gl−1. Furthermore, the flow behaviour of fermentation broth was Newtonian and the maximum μap was 2.7×10−3Pas. The MW of laccase was 64kDa and it showed a pI value of 4.2. The CD analysis showed that laccase had a high α-helical content (68%). The MW of the purified EPS was determined to be 1.8×106Da, consisting of carbohydrates (87.6%) and proteins (12.4%). The EPS consisted of 17 amino acids, mainly serine (11.3%), glutamic acid (12.60%), leucine (13.3%) and phenylalanine (9.4%) in protein moiety, and three monosaccharides (galactose, mannose and xylose).

Characterization of Structure, Dynamics, and Detergent Interactions of the Anti-HIV Chemokine Variant 5P12-RANTES

RANTES (CCL5) is a chemokine that recruits immune cells to inflammatory sites by interacting with the G-protein coupled receptor CCR5, which is also the primary coreceptor used together with CD4 by HIV to enter and infect target cells. Ligands of CCR5, including chemokines and chemokine analogs, are capable of blocking HIV entry, and studies of their structures and interactions with CCR5 will be key to understanding and optimizing HIV inhibition. The RANTES derivative 5P12-RANTES is a highly potent HIV entry inhibitor that is being developed as a topical HIV prevention agent (microbicide). We have characterized the structure and dynamics of 5P12-RANTES by solution NMR. With the exception of the nine flexible N-terminal residues, 5P12-RANTES has the same structure as wild-type RANTES but unlike the wild-type, does not dimerize via its N-terminus. To prepare the ground for interaction studies with detergent-solubilized CCR5, we have also investigated the interaction of RANTES and 5P12-RANTES with various commonly used detergents. Both RANTES variants are stable in Cymal-5, DHPC, Anzergent-3-12, dodecyltrimethylammonium chloride, and a DDM/CHAPS/CHS mixture. Fos-Cholines, dodecyldimethylglycine, and sodium dodecyl-sulfate denature both RANTES variants at low pH, whereas at neutral pH the stability is considerably higher. The onset of Fos-Choline-12-induced denaturation and the denatured state were characterized by circular dichroism and NMR. The detergent interaction starts below the critical micelle concentration at a well-defined mixed hydrophobic/positive surface region of the chemokine, which overlaps with the dimer interface. An increase of Fos-Choline-12 concentration above the critical micelle concentration causes a transition to a denatured state with a high α-helical content.

Heterologous expression and purification of an active human TRPV3 ion channel

The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high-level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We selected a suitable detergent and buffer system using analytical size-exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α-helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies.

Stress fermentation strategies for the production of hyperthermostable superoxide dismutase from Thermus thermophilus HB27: effects of ions

In this study, we explored how ammonium and metal ion stresses affected the production of recombinant hyperthermostable manganese superoxide dismutase (Mn-SOD). To improve Mn-SOD production, fed-batch culture in shake flasks and bioreactor fermentation were undertaken to examine the effects of [Formula: see text] and Mn(2+) feeding. Under the optimized feeding time and concentrations of [Formula: see text] and Mn(2+), the maximal SOD activity obtained from bioreactor fermentation reached some 480U/ml, over 4 times higher than that in batch cultivation (113U/ml), indicating a major enhancement of the concentration of Mn-SOD in the scale-up of hyperthermostable Mn-SOD production. In contrast, when the fed-batch culture with appropriate [Formula: see text] and Mn(2+) feeding was carried out in the same 5-L stirred tank bioreactor, a maximal SOD concentration of some 450U/ml was obtained, again indicating substantial increase in SOD activity as a result of [Formula: see text] and Mn(2+) feeding. The isoelectric point (pI) of the sample was found to be 6.2. It was highly stable at 90°C and circular dichroism measurements indicated a high α-helical content of 70% as well, consistent with known SOD properties. This study indicates that [Formula: see text] and Mn(2+) play important roles in Mn-SOD expression. Stress fermentation strategies established in this study are useful for large-scale efficient production of hyperthermostable Mn-SOD and may also be valuable for the scale-up of other extremozymes.

Oligomerization state of the multimerization domain of Nipah virus phosphoprotein

Oligomerization of the phospho (P) proteins of paramyxoviruses plays important roles in the viral genome replication and transcription. The multimerization domain of the P proteins (PMD) was found to be responsible and sufficient for the oligomerization process. However, the oligomeric status of Nipah virus (NiV) P protein is still unknown. Therefore, the aim of this study was to produce the PMD region of NiV in bacteria in order to determine the oligomeric status of the P protein. The NiV PMD region was cloned into pTrcHis2-TOPO vector and expressed in Escherichia coli. SDS-PAGE and Western blot analysis confirmed the protein expression upon IPTG induction at 37°C for 5h. Immobilized metal affinity chromatography (IMAC) purification was developed to purify the recombinant histidine-tagged NiV PMD which yielded a purity of 97%. Chemical cross-linking revealed that the PMD of NiV formed tetramer. The stability of the oligomer was studied with dynamic light scattering while mass spectrometry confirmed the exact molecular masses of the cross-linked products. Circular dichroism analysis revealed that the NiV PMD contained high α-helical content (55%) suggesting the presence of a coiled coil structure.

SOLVENT INDUCED CHANGES IN THE CONFORMATIONAL STATE OF β-LACTOGLOBULIN AND THE INFLUENCE OF PROTIC IONIC LIQUIDS

The protic ionic liquids (pILs), triethylammonium acetate, triethylammonium trifluoroacetate, triethylammonium mesylate and trimethylammonium sulfate were used to induce various native and non-native conformational states of the protein β-lactoglobulin (βLG). Changes in the secondary structure of βLG were observed on moving from a high water content to a high pIL content. We examined the stability of various pIL induced states via thermal unfolding and refolding, where it was found that at a given pIL concentration a highly stable non-native conformation was formed. The βLG non-native conformation was characterized by a high α-helical content. Additionally, pIL conditions that promoted amyloid fibril formation were identified and characterized by CD, a Thioflavin T binding assay and transmission electron microscopy (TEM). This work highlights the use of pILs as solvents in the study of protein folding using βLG as a model system.