Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

The roles of PGF2α and PGE2 in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0 = day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF_2α (PGFM), 13,14-dihydro-15-keto-PGE₂ (PGEM) and progesterone (P₄) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2 pg/mL (from 36.8 ± 7.8 pg/mL on Day 3 to 163 ± 37.2 pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4 pg/mL (from 128.2 ± 27.8 pg/mL on Day 3 to 326.6 ± 115.1 pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF_2α than by PGE₂, and the increase ratio of PGF_2α was more important than the raw increase.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Visual learning and memory of Drosophila melanogaster wild type CS and the mutants dunce1, amnesiac, turnip and rutabaga

Dunce¹, amnesiac, turnip and rutabaga, mutants of Drosophila melanogaster deficient in olfactory learning and/or memory, were tested for visual learning ability and memory. All these mutants are able to learn conditioned visual information, but not as well as the corresponding wildtype CS. Memory of all four mutants is reduced during the first 30 min after training, but indistinguishable from that of the wildtype two hours after conditioning.     

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.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

Critical evaluation of electron transfer kinetics in P700–FA/FB, P700–FX, and P700–A1 Photosystem I core complexes in liquid and in trehalose glass

This work aims to fully elucidate the effects of a trehalose glassy matrix on electron transfer reactions in cyanobacterial Photosystem I (PS I). Forward and backward electron transfer rates from A_1A^? and A_1B^? to F_X, and charge recombination rates from A₀^?, A_1B^?, A_1A^?, F_X^?, and [F_A/F_B]^? to P₇₀₀⁺ were measured in P₇₀₀–F_A/F_B complexes, P₇₀₀–F_X cores, and P₇₀₀–A₁ cores, both in liquid and in a trehalose glassy matrix at 11% humidity. By comparing CONTIN-resolved kinetic events over 6 orders of time in increasingly simplified versions of PS I at 480?nm, a wavelength that reports primarily A_1A^?/A_1B^? oxidation, and over 9 orders of time at 830?nm, a wavelength that reports P₇₀₀⁺ reduction and A₀^? oxidation, assignments could be made for nearly all of the resolved kinetic phases. Trehalose-embedded PS I samples demonstrated partially arrested forward electron transfer. The fractions of complexes in which electron transfer did not proceed beyond A₀, A₁ and F_X were 53%, 16% and 22%, respectively, with only 10% of electrons reaching the terminal F_A/F_B clusters. The ~10?μs and ~150?μs components in both liquid and trehalose-embedded PS I were assigned to recombination between A_1B^? and P₇₀₀⁺ and between A_1A^? and P₇₀₀⁺, respectively. The kinetics and amplitudes of these resolved kinetic phases in liquid and trehalose-embedded PS I samples could be well-fitted by a kinetic model that allowed us to calculate the asymmetrical contribution of the A_1A^? and A_1B^? quinones to the electrochromic signal at 480?nm. Possible reasons for these effects are discussed.     

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).     

N,O versus O,O coordination in β-imino diketonato complexes: Role of the metal center and of the imino substituent

β-Imino carbonyl enolato metal(II) complexes of general formula [M((RCO)(R′CO)CC(R″)NH)₂] (M = Mn, Fe, Co, Ni, Cu, Zn, Pd, R = R′ = Me, R″ = CCl₃; M = Cu, Pd, R = R′ = Me, R″ = PhCO; R = Me, R′ = Ph, R″ = PhCO; R = R′ = Ph, R″ = PhCO) are easily synthesized by the reaction of metal(II) acetates with the proper β-enaminodiones in 1/2 molar ratio in ethanol at room temperature. In all the cases the trifunctional NOO β-imino carbonyl enolate ligand acts as bidentate to give ML₂ complexes, whose structure depends on the metal center and on the nature of the substituent R″ at the imino carbon. With R″ = CCl₃ an O,O coordination is observed for all the metal centers but one, in fact palladium(II) exhibits an N,O coordination through the imino nitrogen and one keto oxygen. By contrast with R″ = PhCO the ligand is always coordinated through the imino nitrogen and one keto oxygen atom.     

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.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.     

Rôle of the midgut,crop, and maxillae of Bombyx mori in the production of cocoon-digesting enzyme

The rôle of the midgut, crop, and maxillae in the production and utilization of the cocoon-digesting enzyme was investigated in the silkworm, Bombyx mori.About a sixtyfold purified preparation of midgut protease was obtained by ammonium sulphate precipitation and column chromatography.Immunological studies by the agar diffusion method of Ouchterlony revealed that the crop and midgut proteases of the pharate adult are antigenically identical whereas that of the maxillary protease is different.From the results of extirpation experiments and previous studies it was shown that the midgut, crop, and maxillae play important rôles in the escape of moths from their cocoons.     

1,3-Bis(α-aminoisopropyl)benzene, meta-C6H4(CMe2NH2)2: An N,N-bridging and N,C,N-cyclometalating ligand

Saponification of the bis(carbamic acid ester) 1,3-C₆H₄(CMe₂NHCO₂Me)₂ (1), made by the addition of methanol to commercial 1,3-C₆H₄(CMe₂NCO)₂, yielded the meta-phenylene-based bis(tertiary carbinamine) 1,3-C₆H₄(CMe₂NH₂)₂ (2). Dinuclear [{(η⁴-1,5-C₈H₁₂)RhCl}₂{μ-1,3-C₆H₄(CMe₂NH₂)₂}] (3) resulted from the action of 2 on [{(η⁴-1,5-C₈H₁₂)Rh(μ-Cl)}₂] in toluene. Combination of 2 with PdCl₂ or K₂[PdCl₄] gave the dipalladium macrocycle trans,trans-[{μ-1,3-C₆H₄(CMe₂NH₂)₂}₂(PdCl₂)₂] (4) along with cyclometalated [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹,κN′}PdCl] (5). Substitution of PEt₃ for the labile chlorido ligand of 5 afforded [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹, κN′}Pd(PEt₃)]Cl (6). The crystal structures of the following compounds were determined: bis(carbamic acid ester) 1, ligand 2 as its bis(trifluoroacetate) salt [1,3-C₆H₄(CMe₂NH₃)₂](O₂CCF₃)₂, 2 · (HAc_f)₂, complexes 3 and 6, as well as 1,3-C₆H₄(CMe₂OH)₂ (the diol analogue of 2).     

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.