Intermediates I1 Research Articles

Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( ~ 550s-1, ~ 1200s-1, ~ 5000s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15°C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

Synthesis of Lobeglitazone intermediates seeking for continuous drug production in flow capillary microreactor

Continuous flow synthesis in microreactors has been integrated into chemical-pharmaceutical industry in recent years as an alternative to the batch process due to its advantages, especially process intensification, which can reduce the time for a new drug to be placed on the market on a large scale. This work aimed to transpose the synthesis of Lobeglitazone, a drug employed in the treatment of diabetes mellitus type 2, from batch to flow process in a microreactor as well as to determine the reaction kinetics of each step. The synthesis was carried out in five-steps, being synthesized intermediates 4-chloro-6-(4-methoxyphenoxy)pyrimidine (I1), 2-{[6-(4-methoxyphenoxy)pyrimidin-4-yl]methylamino}ethanol (I2), 4-(2-{[6-(4-methoxyphenoxy)pyrimidin-4-yl]methylamino}ethoxy)benzaldehyde (I3), 5-[4-(2-{[6-(4-methoxyphenoxy)pyrimidin-4-yl]methylamino}ethoxy)benzylidene]thiazolidine-2,4-dione (I4) and Lobeglitazone. Intermediates I1 and I4 were synthesized in flow, while I4 was synthesized either in a continuous flow multistep synthesis or in a one-pot batch process. The flow syntheses of I1, I2 and I4 showed 28.0 %, 61.8 % and 32.0 % yields at 25, 160 and 120 °C, respectively, while the yield of I3 in batch process was 73.3 % at 60 °C. In one-pot batch process and continuous flow multistep synthesis, I2 was obtained with 13 and 16 % yields, respectively. These preliminary results constitute a starting point for the synthesis of this drug in flow on an industrial scale, with the aim of improving reaction performance using this new technology.

Probing the Acid-Induced Packing Structure Changes of the Molten Globule Domains of a Protein near Equilibrium Unfolding.

Using simultaneously scanning small-angle X-ray scattering (SAXS) and UV-vis absorption with integrated online size exclusion chromatography, supplemental with molecular dynamics simulations, we unveil the long-postulated global structure evolution of a model multidomain protein bovine serum albumin (BSA) during acid-induced unfolding. Our results differentiate three global packing structures of the three molten globule domains of BSA, forming three intermediates I1, I2, and E along the unfolding pathway. The I1-I2 transition, overlooked in all previous studies, involves mainly coordinated reorientations across interconnected molten globule subdomains, and the transition activates a critical pivot domain opening of the protein for entering into the E form, with an unexpectedly large unfolding free energy change of -9.5 kcal mol-1, extracted based on the observed packing structural changes. The revealed local packing flexibility and rigidity of the molten globule domains in the E form elucidate how collective motions of the molten globule domains profoundly influence the folding-unfolding pathway of a multidomain protein.

Ibuprofen and propofol cobinding effect on human serum albumin unfolding in urea.

The unfolding pathway of the defatted human serum albumin (HSA) binding ibuprofen and propofol has been studied by using small-angle X-ray scattering (SAXS) and the support of circular dichroism data. A set of HSA solutions with urea concentrations between 0.00 and 9.00 M was analyzed, and the singular value decomposition method applied to the complete SAXS data set allowed us to distinguish four different states in solution. Besides the native and unfolded forms, two intermediates I1 and I2 have been identified, and the low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. The I1 structure was characterized by only one open domain (domain I, which does not host a binding site for either of the ligands), whereas I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex (Galantini et al. Biophys. Chem. 2010, 147, 111-122) pointed out that the presence of propofol as a second ligand, located in subdomain IIIB, leads to the appearance of an intermediate with two closed domains (domains II and III), which are those that accommodate the ligands. Moreover, the equilibrium between I2 and the unfolded form is slightly shifted toward higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process.

The Transient Accumulation of the Signaling State of Photoactive Yellow Protein Is Controlled by the External pH

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I2′. The pH dependence of the equilibrium between the transient photocycle intermediates I2 and I2′ was investigated. The formation of I2′ from I2 is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I1, I2, and I2′. Their spectra were determined using the extrapolated difference method. I2 and I2′ have electronic absorption spectra, with maxima at 370±5 and 350±5nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I1, I2, and I2′ intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I2′ (∼2ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I2 and I2′ is pH dependent with a pKa of 6.4 and with I2′ the main species above this pKa. Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355nm at a delay of 20ms confirm this pKa value. I2 and I2′ are photoreversed with reversal times of ∼55μs and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pKa of ∼6.1. Photoreversal from I2′ dominates above the pKa. The transient accumulation of I2′, the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k3 for the recovery to the initial dark state also has a pKa of ∼6.3. This equality of the equilibrium and kinetic pKa values is not accidental and suggests that k3 is proportional to [I2′].

Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy

BackgroundPrions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates.ResultsConformational intermediates of the human prion protein huPrPC were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrPC(121–230) and the full-length huPrPC(23–230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N1and N2 in solution associated with rather small differences in free enthalpies (3.0 kJ/mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N2. There is a second process representing two possible folding intermediates I1 and I2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure.ConclusionWe identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the β1/α1-loop and the solvent exposed side of α3. The most pressure-sensitive region (representing mainly intermediate I1) is the loop between β-strand 1 and α-helix 1 (residue 139–141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein.

Time-Resolved Single Tryptophan Fluorescence in Photoactive Yellow Protein Monitors Changes in the Chromophore Structure during the Photocycle via Energy Transfer

We show from time-resolved fluorescence intensity and depolarization experiments that the fluorescence of the unique tryptophan W119 of PYP is quenched by energy transfer to the 4-hydroxycinnamoyl chromophore. Whereas the intensity decay has a time constant of 0.18 ns in P, the decay in the absence of the cofactor (apo-PYP) has a single exponential lifetime of 4.8 ns. This difference in lifetime with and without acceptor can be explained quantitatively on the basis of energy transfer and the high-resolution X-ray structure of P, which allows an accurate calculation of the kappa2 factor. Fluorescence depolarization experiments with donor and acceptor indicate that both are immobilized so that kappa2 is constant on the fluorescence time scale. Using background illumination from an LED emitting at 470 nm, we measured the time-resolved fluorescence in a photostationary mixture of P and the intermediates I2 and I2'. The composition of the photostationary mixture depends on pH and changes from mainly I2 at low pH to predominantly I2' at high pH. The I2/I2' equilibrium is pH-dependent with a pKa of approximately 6.3. In I2 the lifetime increases to approximately 0.82 ns. This is not due to a change in distance or to the increase in spectral overlap but is primarily a consequence of a large decrease in kappa2. Kappa2 was calculated from the available X-ray structures and decreases from approximately 2.7 in P to 0.27 in I2. This change in kappa2 is caused by the isomerization of the acceptor, which leads to a reorientation of its transition dipole moment. We have here a rare case of the kappa2 factor dominating the change in energy transfer. The fluorescence decay in the light is pH-dependent. From an SVD analysis of the light/dark difference intensity decay at a number of pH values, we identify three species with associated lifetimes: P (0.18 ns), I2 (0.82 ns), and X (0.04 ns). On the basis of the pH dependence of the amplitudes associated with I2 and X, with a pKa of approximately 6.3, we assign the third species to the signaling state I2'. The absorption spectra of the 0.82 and 0.04 ns species were calculated from the pH dependence of their fluorescence amplitudes and of the photostationary light/dark difference absorption spectra. The lambda(max) values of these spectra (372 and 352 nm) identify the 0.82 and 0.04 ns components with I2 and I2', respectively, and validate the fluorescence data analysis. The mutant E46Q allows a further test of the energy transfer explanation, since lowering the pH in the dark leads to a bleached state with an increased spectral overlap but without the isomerization-induced decrease in kappa2. The measured lifetime of 0.04 ns is in excellent agreement with predictions based on energy transfer and the X-ray structure.

Growth of strained Si on high-quality relaxed Si1−xGex with an intermediate Si1−yCy layer

An intermediate Si1−yCy layer in the Si1−xGex film, replacing the conventionally graded buffer layer, was used to form the high-quality relaxed SiGe substrate. With the 700 nm thick Si0.8Ge0.2 overlayer, such a Si1−xGex∕Si1−yCy∕Si1−xGex (x=0.2, y−0.014) heterostructure has a threading dislocation density of 5.5×105cm−2 and a residual strain of only 2%. The surface roughness was measured to be about 4.2 nm. The long-range misfit dislocation array was formed mainly at the interface of top Si1−xGex and Si1−yCy. Strained-Si n-channel metal-oxide-semiconductor transistors with various buffer layers were fabricated and examined. Effective electron mobility for the strained-Si device with this substrate technology was found to be 95% higher than that of Si control device. The scheme for the formation of the relaxed Si1−xGex film serving as a virtual substrate shall be applicable to high-speed strained-Si devices.

Mechanism of an alcohol-trapping reaction in the direct and benzophenone-sensitized photodenitrogenation of a spiroepoxy-substituted azoalkane: solvent effects and stereochemical deuterium labeling.

The spin-state-dependent reactivity, singlet versus triplet, of the 2-spiroepoxy-1,3-cyclopentane-1,3-diyl DR2 has been assessed through alcohol-trapping reactions for which the effect of solvent acidity on the product distribution of the alcohol trapping products 2 versus 3 + 4 and stereochemical deuterium-labeling studies have been performed. The proposed mechanism for the solvent effect on the product ratio (2/3 + 4) reveals the importance of the hydrogen-bonded intermediates I1 and I2 in the trapping reactions; the stereochemical deuterium-labeling results clarify the dipole structure trapped by the alcohol. The dipoles DP1 and DP2, in which the configuration between the epoxide oxygen and the deuterium atoms is retained, are inferred for the direct photodenitrogenation reactions (singlet state), whereas for the benzophenone-sensitized photoreactions (triplet state), after ISC, the ring-opened dipole DP3 is implied as the intermediate that is trapped by the alcohol.

A combined crossed beam and ab initio investigation on the reaction of carbon species with C4H6 isomers. III. 1,2-butadiene, H2CCCH(CH3) (X 1A′)—a non-Rice–Ramsperger–Kassel–Marcus system?

Crossed molecular beam experiments were conducted to investigate the reaction of ground state carbon atoms, C(3Pj), with 1,2-butadiene, H2CCCH(CH3) (X 1A′), at three collision energies of 20.4 kJ mol−1, 37.9 kJ mol−1, and 48.6 kJ mol−1. Ab initio calculations together with our experimental data reveal that the reaction is initiated by a barrier-less addition of the carbon atom to the π system of the 1,2-butadiene molecule. Dominated by large impact parameters, C(3Pj) attacks preferentially the C2–C3 double bond to form i1 (mechanism 1); to a minor extent, small impact parameters lead to an addition of atomic carbon to the C1–C2 bond yielding i2 (mechanism 2). Both cyclic intermediates i1 and i2 ring open to triplet methylbutatriene complexes i3′ (H2CC*CCH(CH3)) and i3″, (H2CCC*CH(CH3)); C* denotes the attacked carbon atom. i3′ is suggested to decay nonstatistically prior to a complete energy randomization via atomic hydrogen loss forming 1- and 4-methylbutatrienyl CH3CCCCH2 (X 2A″) and HCCCCH(CH3) (X 2A″), respectively. The energy randomization in i3″ is likely to be complete. This isomer decomposes via H atom loss to 3-vinylpropargyl, H2CCCC2H3(X 2A″), as well as 1- and 4-methylbutatrienyl radicals. In high-density environments such as the inner regions of circumstellar envelopes of carbon stars and combustion flames, these linear C5H5 isomers might undergo collision induced isomerization to cyclic structures like the cyclopentadienyl radical. This isomer is strongly believed to be a key intermediate involved in the production of polycyclic aromatic hydrocarbon molecules and soot formation. These characteristics make the reactions of atomic carbon with C4H6 isomers compelling candidates to form C5H5 isomers in the outflow of AGB stars and oxygen-deficient hydrocarbon flames.

Growth of perfect-crystal Si-Si1−x Gex-(Ge2)1−x (InP)x structures from the liquid phase

Structures comprising Si-Si1−x Gex-(Ge2)1−x (InP)x with an intermediate Si1−x Gex buffer layer were grown on silicon substrates. Morphological examinations, scanning patterns and diffraction spectra, and also the electrophysical and luminescence properties of the heterostructures were used to show that the crystal perfection of these structures depends on the choice of liquid-phase epitaxy conditions.

Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B

Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.

VLPCVD Heteroepitaxial Growth of Very Thin GE-Layers on Si-Substrates

ABSTRACTBy Pulsed Gas CVD-growth, heteroepitaxial growth of pure Ge-layers from pure GeH4 has been accomplished at 650 °C on Si-substrates without intermediate Si1−xGex-bufferlayers. The material has been studied by means of RBS, SEM, ECP, XRD and cross-sectional, highresolution and plan-view TEM. The thickness of the Ge-layers varied between 2 and 24 monolayers. From 5 monolayers on, some part of the Ge appears to be fully relaxed. Uptill 12 monolayers, the growth is fully 2-dimensional and results in specular, flat surfaces; for thicker layers islanding starts (Stranski-Krastanow growth). The material is completely defect-free. According to XRD-measurements, the perpendicular strained Ge-cell dimension is 5.748 Å, which is very reproducible for all layer-thicknesses. Nevertheless, from linear elastic theory considerations, it should be 5.83 Å. The Ge-layers so formed appear to be suitable for use in short-period (SinGem)p-superlattices.