Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T₁, a denaturant-dependent transition and T₂, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub^M1W, Ub^S57W) display Trp fluorescence changes during T₁ that are distinct from the expected U → N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U → N transition but represents the formation of a structurally distinct I-state (U → I). Alternatively, this U → I transition could be also clearly distinguished by using a combination of two Trp mutations Ub^F45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T₁ and T₂ (Ub^F45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub^M1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.     

Solvent and electrolyte effects in enhancing the identification of intramolecular electronic communication in a multi redox-active diruthenium tetraferrocenoate complex, a triple-sandwiched dicadmium phthalocyanine and a ruthenocene-containing β-diketone

Enhanced electrochemical resolution of anodic processes is possible in the presence of [N(ⁿBu)₄][B(C₆F₅)₄], 1, as supporting electrolyte over that obtained in the presence of [N(ⁿBu)₄][PF₆]. By changing the anion of the supporting electrolyte to a salt having [B(C₆F₅)₄]⁻, anions, electrochemical processes of especially cationic analytes can benefit. Thus, the redox chemistry of 0.5 mmol dm⁻³ solutions of [Ru₂(μ-FcCOO)₄·(CH₃CH₂OH)₂][PF₆], 2, Fc = ferrocenyl, in CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] were found to involve four well-resolved ferrocenyl-based electrochemical reversible redox processes as well as reduction of Ru^III-Ru^II. At 1.0 mmol dm⁻³ concentrations of 2, or in the presence of [N(ⁿBu)₄][PF₆], the four ferrocenyl processes coalesced into only two waves as a result of (Fc⁺)?() ion paring. Seventeen of the possible 18 one-electron transfer processes of the biscadmium trisphthalocyaninato complex [Cd₂{Pc(C₆H₁₃)₈}₃], 3, could be observed in THF/[N(ⁿBu)₄][B(C₆F₅)₄], but the electrochemical window of CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] only allowed detection of 15 of these processes. Although reduction processes were unaffected, THF solvation leading to species such as (3ⁿ⁺)(THF)_x with 1 ? n ? 4 and x ? 1 as well as ion pair formation of the type (3ⁿ⁺)?() prevented good resolution of oxidation processes. The CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] system also allowed detection of reversible one-electron transfer ferrocenyl (Fc/Fc⁺) and ruthenocenyl-based (Rc/Rc⁺) processes for both enol and keto isomers of the β-diketone FcCOCH₂CORc, 4, Rc = ruthenocenyl. In CH₃CN/[N(ⁿBu)₄][PF₆], the ruthenocenyl moiety was oxidised to a Ru^IV species.     

Syntheses, electrolytic behaviour and antifungal activities of Zn(II) complexes of isomers of 3,10-C-meso-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L). Crystal and molecular structure of [ZnLB(NO3)]NO3 (LB = a,e,a,e-L)

The isomeric cyclam ligands Me₈[14]anes, designated by L_A, L_B and L_C, produce, on reaction with zinc(II)nitrate, zinc(II)sulphate or zinc(II)chloride corresponding complexes, i.e. dinitrato/mononitrato-nitrate complexes [ZnL(NO₃)₂]/[ZnL(NO₃)](NO₃) (L = L_A, L_B or L_C, where the indices A, B and C refer to differing orientations of the four methyl groups on secondary carbons of Me₈[14]ane)_, the diaqua-sulphates [ZnL(H₂O)₂]SO₄ (L = L_A, L_B or L_C), and the diaqua dichloride and dichlorido complexes [ZnL(H₂O)₂]Cl₂ (L = L_A or L_C) or [ZnL_BCl₂], respectively. The complexes have been characterised on the basis of elemental analyses, IR, UV-Vis, ¹H and ¹³C NMR spectroscopies, magnetic and conductance data. The structure of [ZnL_B(NO₃)](NO₃) has been determined by X-ray crystallography. The zinc centre is coordinated to a N₄O donor set in a square-pyramidal geometry. The complexes show differing electrolytic behaviour in different solvents. In chloroform, the complexes are non-electrolytes, indicating that both anions are coordinated to Zn²⁺. Antifungal activity of the ligands and complexes against the phytopathogenic fungi Alternaria alternata and Colletotrichum corcolei have been investigated, and positive results were noted.     

Metal-metal stacking patterns between and with [Pt(tpy)X] cations

A comparative study of metallophilic interactions of [Pt(tpy)X]⁺ cations (tpy = 2,2′:6′,2″-terpyridine) in the presence of two different types of anions, (i) []⁻ anions that form double salts and (ii) simple p-block anions, is reported. Single-crystal X-ray diffraction data, solution-state ¹⁹⁵Pt NMR spectra, and variable temperature solid-state luminescence spectra are reported. Three [Pt(tpy)Cl]Y derivatives (Y = SbF₆, 1, SbF₆·CH₃CN, 4, PF₆, 2) and the [Pt(tpy)Br]PF₆ analog, 3, as well as two new double salts [Pt(tpy)CN][Au(CN)₂], 5, and [Pt(tpy)CN]₂[Au(C₆F₅)₂](PF₆), 6, have been synthesized and characterized. Structural analysis shows consistent patterns in Pt···Pt interactions that vary slightly depending on the coordinating halogen or pseudo-halogen X, counter anion Y, and lattice solvent. Metallophilic interactions are seen between [Pt(tpy)X]⁺ cations with all types of X ligands, but only with π-accepting X′ ligands from []⁻ anions are Pt?Au metallophilic interactions seen to be favored over Pt?Pt interactions. The [Au(CN)₂]⁻ anion consistently forms Pt···Au metallophilic contacts, unlike [Au(C₆F₅)₂]⁻. The ¹⁹⁵Pt NMR chemical shifts are ∼−2750 ppm for π-donor ligands and near −3120 ppm for π-acceptor ligands in [Pt(tpy)X]PF₆ compounds. Luminescence data show an unusual blue shift in [Pt(tpy)CCPh][Au(C₆F₅)₂] versus [Pt(tpy)CCPh]PF₆ ascribed to an intermolecular charge transfer.     

The solid-liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids: differing by four carbon atoms

The complete solid-liquid phase diagrams for four binary mixtures of saturated fatty acids are presented, for the first time, in this work. These mixtures are formed by caprylic acid (C_8:0) + lauric acid (C_12:0), capric acid (C_10:0) + myristic acid (C_14:0), lauric acid (C_12:0) + palmitic acid (C_16:0) and myristic acid (C_14:0) + stearic acid (C_18:0). The phase diagrams were obtained by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). FT-Raman spectrometry and polarized light microscopy were used to complement the characterization for a complete understanding of the phase diagram. All of the phase diagrams here reported show the same global behavior that is far more complex than previously accepted. They present not only peritectic and eutectic reactions, but also metatectic reactions, due to solid-solid phase transitions common in fatty acids, and regions of solid solution not previously reported. This work contributes to the elucidation of the phase behavior of these important biochemical molecules with implications in various industrial applications.     

Self-assembly of cationic palladium complexes by redistribution of pyridine ligands

The reaction of [Pd(OAc)₂(py)₂] with [Li((OEt₂)_2.5)][B(C₆F₅)₄] was conducted with intent to generate the cationic palladium complex [Pd(OAc)(py)₃][B(C₆F₅)₄], (2, py = pyridine). A single crystal structure of this material, however, reveals a 1-D polymer structure formed by the self-assembly of alternating dicationic ([Pd(py)₄]²⁺) and neutral ([Pd(OAc)₂(py)₂]) palladium units bridged by acetato linkages to give [Pd(py)₄][Pd(OAc)₂(py)₂][B(C₆F₅)₄]₂ (3). These two palladium sites are produced by disproportionation of the pyridine ligands in [Pd(OAc)(py)₃][B(C₆F₅)₄]. Proton NMR studies confirm the existence of a solvent dependent equilibrium between [Pd(py)₄]²⁺, [Pd(OAc)₂(py)₂] and [Pd(OAc)(py)₃]⁺.     

Cationic copper(I) and silver(I) nitrile complexes with fluorinated weakly coordinating anions: Metal–nitrile bond strength and its influence on the catalytic performance

Copper(I) and silver(I) complexes of formulae [Cu(NCCH₃)₄]⁺[A]⁻ ([A]⁻ = [B(C₆F₅)₄]⁻ (1), {B[C₆H₃(CF₃)₂]₄}⁻ (2), [(C₆F₅)₃B–C₃H₃N₂–B(C₆F₅)₃]⁻ (3), and [Ag(NCCH₃)₄]⁺[B(C₆F₅)₄]⁻ (4) are examined with particular emphasis on the strength of their M–N bond and its influence on the catalytic performance of these complexes in cyclopropanation and aziridination. To examine the strength of the M–N interactions, vibrational spectra of the related hydrogenated and deuterated species [Cu(NCCH₃)₄]⁺, [Cu(NCCD₃)₄]⁺, [Ag(NCCH₃)₄]⁺, and [Ag(NCCD₃)₄]⁺ are also determined. It is found that the metal–nitrile bond strength is an important factor for the catalytic activity of the respective complexes.     

Metal-free carbon dioxide reduction and acidic C-H activations using a frustrated Lewis pair

Activation of CO₂ and acidic C-H bonds by the lutidine-tris(pentafluorophenyl)borane [Lut/B(C₆F₅)₃] frustrated Lewis pair (FLP) are described (lutidine = 2,6-dimethylpyridine). Lut/B(C₆F₅)₃ reacts with CO₂ and H₂ at ambient temperature and 4 atm of pressure to form the lutidinium boro-formate salt [LutH⁺][HC(O)OB(C₆F₅)₃⁻]. This salt has been fully characterized including an X-ray crystal structure and independent synthesis from formic acid and Lut/B(C₆F₅)₃. Attempts to activate a C-H bond in methane by Lut/B(C₆F₅)₃, analogous to its heterolytic cleavage of H₂, were unsuccessful, which are consistent with published calculations showing significant barriers to this reaction. Lut/B(C₆F₅)₃ does react with more acidic C-H bonds, including acetone and nitroalkanes. With nitromethane, the boro-nitrone anion H₂CNO₂B(C₆F₅)₃⁻ is formed, as indicated by NMR and mass spectral analyses.     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.     

Synthesis and characterization of ruthenium and osmium complexes of heterocyclic bidentate ligands (N, X), X = S, Se

The reaction of [RuCl₂(PPh₃)₃] and [OsBr₂(PPh₃)₃] precursors with a series of heterocyclic bidentate (N, X) ligands, X = S, Se, gave complexes [M(R-pyS)₂(PPh₃)₂], (R = H, 3-CF₃, 5-CF₃, 3-Me₃Si); [M(R-pymS)₂(PPh₃)₂], (R = 4-CF₃, 4,6-MeCF₃) and [M(R-pySe)₂(PPh₃)₂], (R = H, 3-CF₃, 5-CF₃), where M is Ru or Os, pyS and pymS the anions of pyridine-2-thione and pyrimidine-2-thione, respectively, and pySe is the anion produced by the reductive cleavage of the Se-Se bond in the dipyridyl-2,2′-diselenide. All of the compounds obtained were characterized by microanalysis, IR, FAB, NMR spectroscopy and by cyclic voltammetry. Compounds [Ru(3-CF₃-pyS)₂(PPh₃)₂] · 2(CH₂Cl₂) (2), [Ru(3-Me₃Si-pyS)₂(PPh₃)₂] (4), [Ru(4-CF₃-pymS)₂(PPh₃)₂] (5), [Ru(3-CF₃-pySe)₂(PPh₃)₂] · 2(CH₂Cl₂) (8), [Os(3-CF₃-pyS)₂(PPh₃)₂] · (CHCl₃) (11), [Os(3-Me₃Si-pyS)₂(PPh₃)₂] (13), [Os(3-CF₃-pySe)₂(PPh₃)₂] · 2(CH₂Cl₂) (17), [Os(5-CF₃-pySe)₂(PPh₃)₂] · 2(H₂O) (18) and [OsCl₂(4,6-MeCF₃-pymS)(PPh₃)₂] (19) were also characterized by X-ray diffraction. In all cases, the metal is in a distorted octahedral environment with the heterocyclic ligand acting as a bidentate (N, S) chelate system.     

Analysis of the main structural trends for biscyclometalated dinuclear rhodium compounds of general formula Rh2(O2CR)2(PC)2 · 2H2O

A new series of biscyclometalated dinuclear rhodium (II) compounds with the general formula Rh₂(O₂CR)₂(PC)₂ · 2H₂O, being PC = (C₆H₄)P(C₆H₅)₂, R = CH₃ (1 · 2H₂O), PC = [(p-CH₃ OC₆H₃)P(p-CH₃ OC₆H₄)₂], R = CF₃ (2 · 2H₂O), PC = (C₆H₄)P[CH(CH₃)₂]₂, R = CH₃ (3 · 2H₂O) and PC = (C₆H₄)P(C₆H₅)₂, R = C₆F₅ (4 · 2H₂O) has been obtained. The crystal structures for these compounds have been determined by X-ray diffraction and the main structural trends, bond lengths, bond angles and torsion angles have been analyzed, and have also been compared with the structural parameters for different analogous complexes described previously in the literature.     

Magneto-structural relationships for a mononuclear Co(II) complex with large zero-field splitting

A mononuclear cobalt(II) complex, [Co(ac)₂(H₂O)₂(MeIm)₂], with heteroleptic coordination sphere possessing the {CoO₂O′₂N₂} chromophore has been prepared and structurally characterized. The magnetic data down to 2 K show an enhanced magnetic anisotropy manifesting itself in a large zero-field splitting (ZFS) parameter. As a consequence, the magnetization deviates substantially from the Brillouin-function behavior. A fit to the zero-field splitting model gave the following set of magnetic parameters: D/hc = +95 cm⁻¹, g_x = 2.530, zj/hc = −0.078, χ_TIP = 16.7 × 10⁻⁹ m³ mol⁻¹, (g_z = 2.0). The Griffith-Figgis model and the Generalized Crystal-Field model lie beyond the spin-Hamiltonian formalism; they gave analogous, although not identical ZFS parameters: D/hc = 109 cm⁻¹, and D/hc = 77 cm⁻¹, respectively. The absorption spectrum taken in the FAR-IR region exhibits manifold absorption peaks referring to the transitions among the crystal-field multiplets of the parent ⁴A_2g + ⁴E_g terms (D_4h), originating in a crystal-field splitting of the octahedral ⁴T_1g ground term.     

New phospholyl complexes of groups 4 and 6: Syntheses, characterisation and polymerisation studies

[M(P₃C₂^tBu₂)(CO)₃I] (M = Mo, 1, W, 2) have been synthesised and reacted with PCl₅ for oxidation study purposes. Compounds Ti(P₃C₂^tBu₂)(Ind)Cl₂], 3, and [Zr(P₃C₂^tBu₂)(Cp)Cl₂], 4, were detected spectroscopically, but showed to be too unstable to be isolated. A Ti(IV) complex, [Ti(P₃C₂^tBu₂)Cl₃], 5, has been formed from the reaction of [TiCl₄] with the base-free ligand K(P₃C₂^tBu₂), while the Ti(III) species, [Ti(P₃C₂^tBu₂) Cl₂(THF)], 6, was prepared from [TiCl₃(THF)₃]. Compounds 5 and 6 were studied as ethylene catalyst precursors after activation with MAO. In the studied conditions, complex 5 is the most active one with an activity of 2.2 × 10⁵ g(molTi [E] h)⁻¹, one order of magnitude higher than compound 6. The produced polymer is linear polyethylene.     

Photorespiration and photoprotection of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Cabernet Sauvignon) under water stress

In order to investigate the photoprotective function of photorespiration in grapevine under water stress, potted grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) were randomly divided into three uniform groups for well-watered [watered every morning to keep the relative water content (RWC) of soil over 70 %], water-stress adapted (drought-adapted at 30 % relative soil water content for 30 days), and water stress without adaptation treatment (water-stressed to 30 % relative soil water content for 3 days). Net assimilation rate (A _N), stomatal conductance (g _s), substomatal CO₂ concentration (C _i), transpiration rate (E), actual photochemical efficiency of PSII (Φ_PSII), and maximum photochemical efficiency of PSII (F_v/F_m) were recorded by combining measurements of gas exchange and chlorophyll fluorescence. Gross photorespiration (P_r), photosynthetic electron partitioning (J_C/J_T), photochemical quenching coefficient (qP), and non-photochemical quenching (NPQ) were also calculated. The ratio of net assimilation rate to transpiration rate (A _N/E) was used as an indicator of water use efficiency (WUE). A _N, apparent P_r, Φ_PSII, F_v/F_m, qp, and g _s decreased, NPQ increased, and gross P_r sustained at a high level under water stress. This suggests that both photorespiration and energy dissipation play important roles in protecting photosynthetic apparatus against photoinhibition. C _i in water-stressed plants without adaptation treatment increased, which indicates the leaves suffered a non-stomatal limitation, while the water-stress adaped plants only suffered a stomatal limitation indicated by low C _i.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Crystal structure and magnetic behavior of decamethylferrocenium bis(2-thioxo-1,3-dithiole-4,5-diselenolato)nickelate(III)

The synthesis, crystal structure and magnetic properties of the charge transfer salt [Fe(Cp^∗)₂][Ni(dsit)₂], dsit = (2-thioxo-1,3-dithiole-4,5-diselenolato) are described. The crystal structure consists of layers composed by parallel chains, where side-by-side pairs of cations, alternate with face-to-face pairs of anions, ?D⁺D⁺A⁻A⁻D⁺D⁺A⁻A⁻?. The acceptors in the AA pairs are strongly dimerized through a Ni-Se bond. Short interdimer contacts (Se-Se and S-S) give rise to a porous 3D network, and the [Fe(Cp^∗)₂]⁺pairs are located in the cavities of the acceptors network. The [Fe(Cp^∗)₂]⁺ donors are relatively isolated leading to weak DA and DD interactions and the magnetic behavior of this compound can be basically described as the sum of two contributions: one from nearly independent donors, and the second from the acceptors network, with strong AA AFM coupling, both intradimer and between dimers in adjacent chains.     

Diferrocenes containing thiadiazole connectivities

2,5-Diferrocenyl-1,3,4-thiadiazole, 2,5-Fc₂-^cC₂N₂S, (3) has been synthesized by a two-fold Negishi ferrocenylation of dibromothiadiazole (1) with FcZnCl (2) (Fc = Fe(η⁵-C₅H₄)(η⁵-C₅H₅)) in presence of [Pd(Ph₃P)₄] as catalyst. Additional spacer units between the ferrocenyls and the ^cC₂N₂S core could be introduced by using the Sonogashira C,C cross-coupling protocol. Reaction of 2,5-Br₂-^cC₂N₂S (1) or 2,5-(C₆H₄-4′-I)₂-^cC₂N₂S (6) with FcCCH (4) using [PdCl₂(Ph₃P)₂] and [CuI] as catalyst produced the appropriate organometallics 2,5-(FcCC)₂-^cC₂N₂S (5) or 2,5-(C₆H₄-4′-CCFc)₂-^cC₂N₂S (7). The electronic and structural properties of 3, 5, and 7 were investigated with UV-Vis spectroscopy and single crystal X-ray diffraction (3). Complex 3 adopts a solid state structure with none of the ferrocenyl substituents being coplanar with the thiadiazole ring. Cyclic, square wave, linear sweep voltammetry and in-situ NIR spectro-electrochemistry highlight the electrochemical properties of 3. In dichloromethane (0.1 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄]), compound 3 displays two well resolved electrochemical reversible one-electron events with formal reduction potentials of 0.192 and 0.338 V versus FcH/FcH⁺. In contrast, in presence of [N(ⁿBu)₄][PF₆], the thiadiazoles 3 (E⁰ = 0.22 V), 5 (E⁰ = 0.18 V) and 7 (E⁰ = 0.09 V) show simultaneously oxidation of the two ferrocenyl termini versus FcH/FcH⁺. Spectro-electrochemical studies, performed in a dichloromethane solution of 0.2 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄], also show that 3 can successively be oxidized via 3⁺ to 3²⁺. A weak IVCT absorption (ε ca. 300 L mol⁻¹ cm⁻¹) at 1560 nm was found and is consistent with appreciable interactions between neutral ferrocenyl and positively charged ferrocenium mixed valent intermediates. Mixed-valent compound 3⁺ corresponds to a class II molecule according to Robin and Day.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Genetic association of ADIPOQ gene variants with type 2 diabetes,obesity and serum adiponectin levels in south Indian population

Objective

To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population.

Methods

The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies.

Results

Of the 8 variants, four SNPs namely, + 276 G/T (rs1501299), − 4522 C/T (rs822393), − 11365 C/G (rs266729), and + 712 G/A (rs3774261) were significantly associated with T2DM in our study population. The −3971 A/G (rs822396) and − 11391 G/A (rs17300539) SNPs' association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent association of + 276 G/T (rs1501299) and − 3971 A/G (rs822396) SNPs with generalized obesity and + 349 A/G (rs2241767) with central obesity. Four SNPs, −3971 A/G (rs822396), + 276 G/T (rs1501299), − 4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population.

Conclusion

The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indian population.