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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

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.     

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit,Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca²⁺ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca²⁺ release and Ca²⁺ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca²⁺ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca²⁺ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.     

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.     

A Model of Action Potentials and Fast Ca Dynamics in Pancreatic β-Cells

We examined the ionic mechanisms mediating depolarization-induced spike activity in pancreatic β-cells. We formulated a Hodgkin-Huxley-type ionic model for the action potential (AP) in these cells based on voltage- and current-clamp results together with measurements of Ca²⁺ dynamics in wild-type and Kv2.1 null mouse islets. The model contains an L-type Ca²⁺ current, a “rapid” delayed-rectifier K⁺ current, a small slowly-activated K⁺ current, a Ca²⁺-activated K⁺ current, an ATP-sensitive K⁺ current, a plasma membrane calcium-pump current and a Na⁺ background current. This model, coupled with an equation describing intracellular Ca²⁺ homeostasis, replicates β-cell AP and Ca²⁺ changes during one glucose-induced spontaneous spike, the effects of blocking K⁺ currents with different inhibitors, and specific complex spike in mouse islets lacking Kv2.1 channels. The currents with voltage-independent gating variables can also be responsible for burst behavior. Original features of this model include new equations for L-type Ca²⁺ current, assessment of the role of rapid delayed-rectifier K⁺ current, and Ca²⁺-activated K⁺ currents, demonstrating the important roles of the Ca²⁺-pump and background currents in the APs and bursts. This model provides acceptable fits to voltage-clamp, AP, and Ca²⁺ concentration data based on in silico analysis.     

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.     

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).     

Thermodynamics of β-Sheet Formation in Polyglutamine

The role of β-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of β-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, β-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific β-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of β-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming β-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that β-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.     

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.     

Flow-Induced β-Hairpin Folding of the Glycoprotein Ibα β-Switch

Flow-induced shear has been identified as a regulatory driving force in blood clotting. Shear induces β-hairpin folding of the glycoprotein Ibα β-switch which increases affinity for binding to the von Willebrand factor, a key step in blood clot formation and wound healing. Through 2.1-μs molecular dynamics simulations, we investigate the kinetics of flow-induced β-hairpin folding. Simulations sampling different flow velocities reveal that under flow, β-hairpin folding is initiated by hydrophobic collapse, followed by interstrand hydrogen-bond formation and turn formation. Adaptive biasing force simulations are employed to determine the free energy required for extending the unfolded β-switch from a loop to an elongated state. Lattice and freely jointed chain models illustrate how the folding rate depends on the entropic and enthalpic energy, the latter controlled by flow. The results reveal that the free energy landscape of the β-switch has two stable conformations imprinted on it, namely, loop and hairpin—with flow inducing a transition between the two.