Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

δN and δC analysis of a Posidonia sinuosa seagrass bed

Potential food sources and dominant invertebrates and fishes were collected for the examination of variability in ¹³C/¹²C and ¹⁵N/¹⁴N to determine the sources of carbon available to consumers within a Western Australian Posidonia sinuosa-dominated seagrass bed. Autotrophs showed a wide distribution of δ¹³C values, with P. sinuosa at −11.3 ± 0.8‰ and macroalgae ranging from −16.6 to −31.7‰. This variation allowed us to successfully identify macroalgae as the main contributor of carbon to the trophic structure, although no distinction could be made between epiphytic macroalgae on seagrass, or allochthonous macroalgal sources. The range in δ¹⁵N ratios among potential food items at the trophic base was too small to make it useful as tracer of nitrogen flow pathways, but it consistently increased from macrophytes and detritus (4.1–6.8‰), to invertebrates (5.7–7.4‰) located near the middle of the food web, to fishes (8.3–11.9‰), with piscivorous species such as Leviprora inops generally having a higher ¹⁵N. δ¹³C of seston (−12.8‰) and sedimentary organic matter (−8.7‰) indicate that seagrass material is the main contributor to these two carbon pools, and that very little of it contributes to animal biomass.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Synthesis and characterisation of κ-P and κ-P,N palladium(II) complexes of the open cage water soluble aminophosphine PTN

New palladium(II) complexes containing the water soluble aminophosphine PTN ligand (PTN = 7-phospha-3,7-dimethyl-1,3,5-triazabicyclo[3.3.1]nonane) in 1:1 and 1:2 ratio Pd/PTN ligand, respectively, were prepared and fully characterised by mono and bidimensional ³¹P, ¹H and ¹³C NMR techniques showing that PTN can adopt both κ¹-P and κ²-P,N coordination modes. The complexes with Pd/PTN ratio 1:2 are highly soluble in water at room temperature. Suitable crystals for X-ray structure determination were obtained for the neutral complex κ²-P,N-Pd(PTN)(OAc)₂ (1) and for the monocationic complex [Pd(κ²-P,N-PTN)(κ¹-P-PTN)Cl][PF₆] (5).     

Experimental analysis of the ‘Paarkultureffekt’ in the protandric polychaete, Ophryotrocha puerilis Clap. Mecz.

Female Ophyrotrocha puerilis Clap. Mecz. were coupled. Certain parts of the prostomium and of the pygidium in one or both partners of a couple were amputated in order to prove that they are responsible for the mutual influence which partners normally exert on their sexual differentiation. The results demonstrate that the palps and the ventrolateral prostomial cirri are the transmitters and the ciliated lateral pits on the prostomium the receivers of the mutual influence. The antennae and the pygidial cirri are not necessary as far as the ‘Paarkultureffekt’ is concerned. The nature of the stimulus is still unknown. At the present stage of these investigations, the favoured conception is of a pheromone transmitted during the contact of the partners.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

Variation among diets in discrimination of δC and δN in the amphipod Allorchestes compressa

Discrimination of stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) was examined for the amphipod Allorchestes compressa Dana using controlled laboratory experiments. Amphipods were fed exclusively on single diets (fresh or decomposed macroalgae or seagrass) for three weeks. Macrophyte type (i.e. seagrass, brown algae or red algae) had a greater influence on the stable isotope ratios of A. compressa than the state of decomposition of the macrophyte material. The experiments revealed that δ¹³C in A. compressa stabilised at values lower than those of the diets, which contrasts to the general assumption that consumer-diet discrimination of δ¹³C ranges from 0 to + 1‰. Amphipods fed on seagrass yielded the lowest δ¹³C values, which were 9 to 10‰ lower than their diet, while amphipods fed on macroalgae had values 2 to 4‰ lower than their diet. In addition, contrary to the general assumption that consumer-diet discrimination of δ¹⁵N ranges from + 3 to + 5‰, discrimination of δ¹⁵N was as low as − 1 and + 1 when A. compressa was fed on brown and red algae, respectively, but as high as + 3‰ when fed on seagrass. The results show that discrimination of stable isotopes of carbon and nitrogen can vary considerably depending on the food source, demonstrating that validation of assumptions about discrimination are critical for interpreting stable isotope data from field studies.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Effect of 6α,7β-dihydroxyvouacapan-17β-oic acid and its lactone derivatives on the growth of human cancer cells

The furanditerpene 6α,7β-dihydroxyvouacapan-17β-oic acid (1) is a natural product biosynthesized by some species from the genus Pterodon (Leguminosae). This secondary metabolite has multiple biological activities that include anti-inflammatory, analgesic, plant growth regulatory, anti-edematogenic, photosystem II inhibitory and photosynthesis uncoupler, and antifungal properties. However, few studies on the antiproliferative profile of compound 1 and/or its derivatives have been reported up to date. Here, we describe the isolation of compound 1 from hexane extract of P. polygalaeflorus fruits as well as the semisynthesis of three lactone derivatives: 6α-hydroxyvouacapan-7β,17β-lactone (2), 6α-acetoxyvouacapan-7β,17β-lactone (3), and 6-oxovouacapan-7β,17β-lactone (4). Additionally, antiproliferative activity of these compounds against nine human cancer cell lines was investigated. Our results revealed that 6α-hydroxyvouacapan-7β,17β-lactone (2) was the most potent furanditerpene against all cancer cell lines studied. The presence of non-substituted hydroxyl group at C-6 and the presence of 7β,17β-lactone ring are important for the antiproliferative activity of these compounds.     

Folding Simulations of a De Novo Designed Protein with a βαβ Fold

βαβ structural motifs are commonly used building blocks in protein structures containing parallel β-sheets. However, to our knowledge, no stand-alone βαβ structure has been observed in nature to date. Recently, for the first time that we know of, a small protein with an independent βαβ structure (DS119) was successfully designed in our laboratory. To understand the folding mechanism of DS119, in the study described here, we carried out all-atom molecular dynamics and coarse-grained simulations to investigate its folding pathways and energy landscape. From all-atom simulations, we successfully observed the folding event and got a stable folded structure with a minimal root mean-square deviation of 2.6 Å with respect to the NMR structure. The folding process can be described as a fast collapse phase followed by rapid formation of the central helix, and then slow formation of a parallel β-sheet. By using a native-centric Gō-like model, the cooperativity of the system was characterized in terms of the calorimetric criterion, sigmoidal transitions, conformation distribution shifts, and free-energy profiles. DS119 was found to be an incipient downhill folder that folds more cooperatively than a downhill folder, but less cooperatively than a two-state folder. This may reflect the balance between the two structural elements of DS119: the rapidly formed α-helix and the slowly formed parallel β-sheet. Folding times estimated from both the all-atom simulations and the coarse-grained model were at microsecond level, making DS119 another fast folder. Compared to fast folders reported previously, DS119 is, to the best of our knowledge, the first that exhibits a parallel β-sheet.     

Altered Backbone and Side-Chain Interactions Result in Route Heterogeneity during the Folding of Interleukin-1β (IL-1β)

Deletion of the β-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1β into antagonist activity. Conversely, circular permutations of IL-1β conserve the functional folding route as well as the agonist activity. These two extremes in the folding-functional interplay beg the question of whether mutations in IL-1β would result in changes in the populations of heterogeneous folding routes and the signaling activity. A series of topologically equivalent water-mediated β-strand bridging interactions within the pseudosymmetric β-trefoil fold of IL-1β highlight the backbone water interactions that stabilize the secondary and tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape of IL-1β. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the population of folding routes accessed during folding and signaling activity.     

Ion-Dependent Dynamics of DNA Ejections for Bacteriophage λ

We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage λ. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.     

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III_9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities.     

On the Activation of Integrin αIIbβ3: Outside-in and Inside-out Pathways

Integrin αIIbβ3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin αIIbβ3 is widely known to regulate the process of thrombosis via activation at its cytoplasmic side by talin and interaction with the soluble fibrinogen. It is also reported that three groups of interactions restrain integrin family members in the inactive state, including a set of salt bridges on the cytoplasmic side of the transmembrane domain of the integrin α- and β-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the α- and β-subunits that is known as the outer membrane clasp, and the key interaction group of the βA domain (located on the β-subunit head domain) with the βTD (proximal to the plasma membrane on the β-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the βTD and βA domains have remained ambiguous. In this study, we use molecular dynamics models to take a comprehensive outside-in and inside-out approach at exploring how integrin αIIbβ3 is activated. First, we show that talin’s interaction with the membrane-proximal and membrane-distal regions of integrin cytoplasmic-transmembrane domains significantly loosens the inner membrane clasp. Talin also interacts with an additional salt bridge (R734-E1006), which facilitates integrin activation through the separation of the integrin’s α- and β-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin αIIbβ3. Finally, we show that the interaction of the Arg of the RGD sequence may activate integrin via disrupting the key interaction group between K350 on the βA domain and S673/S674 on the βTD.     

Detergent-Like Activity and α-Helical Structure of Warnericin RK, an Anti-Legionella Peptide

Warnericin RK is the first antimicrobial peptide known to be active against Legionella pneumophila, a pathogen bacterium that is responsible for severe pneumonia. Strikingly, this peptide displays a very narrow range of antimicrobial activity, almost limited to the Legionella genus, and a hemolytic activity. A similar activity has been described for δ-lysin, a well-known hemolytic peptide of Staphylococci that has not been described as antimicrobial. In this study we aimed to understand the mode of action of warnericin RK and to explain its particular target specificity. We found that warnericin RK permeabilizes artificial membranes in a voltage-independent manner. Osmotic protection experiments on erythrocytes showed that warnericin RK does not form well-defined pores, suggesting a detergent-like mode of action, as previously described for δ-lysin at high concentrations. Warnericin RK also permeabilized Legionella cells, and these cells displayed a high sensitivity to detergents. Depending on the detergent used, Legionella was from 10- to 1000-fold more sensitive than the other bacteria tested. Finally, the structure of warnericin RK was investigated by means of circular dichroism and NMR spectroscopy. The peptide adopted an amphiphilic α-helical structure, consistent with the proposed mode of action. We conclude that the specificity of warnericin RK toward Legionella results from both the detergent-like mode of action of the peptide and the high sensitivity of these bacteria to detergents.     

Simultaneous determination of ΔG, ΔH and ΔS by an automatic microcalorimetric titration technique. Application to protein ligand binding

A methodological study has been made with a syringe titration unit attached to an LKB batch microcalorimeter. The presicion and accuracy of the instrument assembly have been evaluated by neutralization reactions and by dilution of sucrose solutions. As an example, heat quantities on the order of 10 mJ accompanying the addition of 10 μl titrant solution could be determined with an accuracy of better than 1%. A stepwise titration procedure was used to characterize the binding of indole-3-propionic acid to α-chymotrypsin. The following thermodynamic data were obtained (25°C, acetate buffer, pH 5.80): ΔG⁰ = ?18.46±0.17 kJ·mol^?1, ΔH⁰ = ?15.26±0.20 kJ·mol^?1, ΔS⁰ = 10.85±1.21 JK^?·mol^?1.     

β1,4-Galactosyltransferase V regulates self-renewal of glioma-initiating cell

Glioma results from unregulated expansion of a self-renewing glioma-initiating cell population. The regulatory pathways which are essential for sustaining the self-renewal of glioma-initiating cells remain largely unknown. Cell surface N-linked oligosaccharides play functional roles in determining cell fate and are associated with glioma malignancy. Previously, we have reported that β1,4-galactosyltransferase V (β1,4GalT V) effectively galactosylates the GlcNAcβ1→6Man arm of the highly branched N-glycans and positively regulates glioma cell growth. Here, we show that decreasing the expression of β1,4GalT V by RNA interference in glioma cells attenuated the formation of polylactosamine and inhibited the ability of tumor formation in vivo. Down-regulation of β1,4GalT V depleted CD133-positive cells in glioma xenograft, and inhibited the self-renewal capacity and the tumorigenic potential of glioma-initiating cells. These data reveal a critical role of β1,4GalT V in the self-renewal and tumorigenicity of glioma-initiating cells, and indicate that manipulating β1,4GalT V expression may have therapeutic potential for the treatment of malignant glioma.     

The segregation of lambs into ‘responders’ and ‘non-responders’: Response to vaccination with irradiated Trichostrongylus colubriformis larvae before weaning

Random bred Merino ram and ewe lambs were vaccinated at 1, 2 and/or 3 months of age with irradiated T. colubriformis larvae. An exponentially increasing challenge of normal larvae was given to all groups including unvaccinated controls commencing at 1 month of age. The results, based on faecal egg counts, showed a dissociation into animals which responded to vaccination (geometric mean egg count 441) and those which did not (geometric mean egg count 1567). The proportion of responders was greatest in groups first vaccinated at the earliest age (1 month). Wool growth and liveweight gains showed severe depression corresponding to peak egg counts, however, responders were less affected than non-responders. There was no correlation between haemoglobin type and resistance to challenge. Faecal egg counts after impulse challenge with 10,000 normal larvae given at about 6$\text{1}\text{2}$ months of age showed a significant ranked correlation with those obtained during the primary exponential challenge. These results confirm that a proportion of young lambs respond to vaccination with irradiated larvae, and that genetically-determined factors are implicated in the ability of animals to respond to vaccination at an early age.