Two-Dimensional Detergent Expansion Strategy for Membrane Protein Studies

Detergents are the most frequently applied reagents in membrane protein (MP) studies. The limited diversity of one-head-one-tailed traditional detergents, however, is far from sufficient for structurally distinct MPs. Expansion of detergent repertoire has a continuous momentum. In line with the speculation that detergent pre-assembly exerts superiority, herein we report for the first time cross-conjugation of two series of monomeric detergents for constructing a two-dimensional library of dimeric detergents. Optimum detergents stood out with unique preferences in the systematic evaluation of individual MPs. Furthermore, unprecedented hybrid detergents 14M8G and 14M9G enabled high-quality EM study of transporter MsbA and NMR study of G protein-coupled receptor A_2AAR, respectively. Given the abundance of cross-coupling chemistries, comprehensive diversity could be readily covered that would facilitate the finding of new detergents for the manipulation of thorny MPs and innovation of the functional and structural study in future.     

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d -maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor, and its complex with G_s protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.     

细胞色素ｃ氧化酶ＣｕＡ结构域蛋白的荧光猝灭研究

近年来 ,有关细胞色素 c 氧化酶的研究取得了重要进展 [1] 。细胞色素 c 氧化酶是由多个亚基组成的复合酶分子 ,其核心亚基Ⅱ中含有可溶性 Cu A 结构域蛋白 ,它是一个高度离域化的混价双核金属中心结构 [Cu 1.5 -Cu 1.5 ],两个铜离子之间由两个 Cys-S - 桥联起来 ,这是生物体     

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction,Overstabilization, and Crystallization of Membrane Proteins

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b , 8 c , and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a , 8 b , 8 f , 9 a , and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A_2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.     

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction,Overstabilization, and Crystallization of Membrane Proteins

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b , 8 c , and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a , 8 b , 8 f , 9 a , and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A_2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.     

pKA in proteins solving the Poisson–Boltzmann equation with finite elements

Knowledge on pK_A values is an eminent factor to understand the function of proteins in living systems. We present a novel approach demonstrating that the finite element (FE) method of solving the linearized Poisson–Boltzmann equation (lPBE) can successfully be used to compute pK_A values in proteins with high accuracy as a possible replacement to finite difference (FD) method. For this purpose, we implemented the software molecular Finite Element Solver (mFES) in the framework of the Karlsberg+ program to compute pK_A values. This work focuses on a comparison between pK_A computations obtained with the well‐established FD method and with the new developed FE method mFES, solving the lPBE using protein crystal structures without conformational changes. Accurate and coarse model systems are set up with mFES using a similar number of unknowns compared with the FD method. Our FE method delivers results for computations of pK_A values and interaction energies of titratable groups, which are comparable in accuracy. We introduce different thermodynamic cycles to evaluate pK_A values and we show for the FE method how different parameters influence the accuracy of computed pK_A values. © 2015 Wiley Periodicals, Inc.     

Control of the Electronic Ground State on an Electron‐Transfer Copper Site by Second‐Sphere Perturbations

The Cu_A center is a dinuclear copper site that serves as an optimized hub for long‐range electron transfer in heme–copper terminal oxidases. Its electronic structure can be described in terms of a σ_u* ground‐state wavefunction with an alternative, less populated ground state of π_u symmetry, which is thermally accessible. It is now shown that second‐sphere mutations in the Cu_A containing subunit of Thermus thermophilus ba₃ oxidase perturb the electronic structure, which leads to a substantial increase in the population of the π_u state, as shown by different spectroscopic methods. This perturbation does not affect the redox potential of the metal site, and despite an increase in the reorganization energy, it is not detrimental to the electron‐transfer kinetics. The mutations were achieved by replacing the loops that are involved in protein–protein interactions with cytochrome c, suggesting that transient protein binding could also elicit ground‐state switching in the oxidase, which enables alternative electron‐transfer pathways.     

Shrinkage and retractive forces during the annealing of drawn low-density polyethylene

Shrinkage of unconstrained low-density polyethylene samples and the retractive stress of samples with ends fixed have been investigated as a function of the annealing time t_A and temperature T_A on material drawn at room temperature to draw ratios λ between 4 and 6. The shrinkage increases with t_A and T_A. The retractive stress on a sample annealed with ends fixed goes through a maximum as the sample is annealed and then drops to a limiting value which increases with T_A as long as T_A is at or below 80°C and rapidly decreases with higher T_A. The drop from the maximum to the limiting retractive stress, slow at lower T_A and rapid at higher T_A, seems to be a consequence of rapid pulling of chain segments out of crystal block in which interfibrillar tie molecules are anchored. This process is facilitated by the high T_A, which softens the crystal matrix. At constant end-to-end distance, the contour length of the tie molecules is irreversibly increased, and this causes a reduction in the contribution of the affected tie molecules to the overall retraction stress. Hence one finds a substantially higher retraction stress during first heating than during subsequent cooling and heating of the drawn sample.     

HETEROGENEITY OF THE QUINONE ELECTRON ACCEPTOR SYSTEM IN BACTERIAL REACTION CENTERS

Abstract— In reaction centers from Rhodopseudomonas viridis, biphasicity of the charge recombination kinetics between P⁺, the primary electron donor, and Q_A and Q_B^-, the primary and secondary quinone electron acceptors, respectively, have been analyzed by the flash-induced absorption change technique. We have studied the effect of quinone environment modifications on the ratio of the two phases for the P⁺Q_A- ([A_fast/A_slow]^a) and P⁺Q_B^- ([A_fast/A_slow]^b) charge recombination processes. In reaction centers from Rps. viridis reconstituted in phosphatidylcholine liposomes a notable influence of the nature of the Q_B pocket occupancy was observed on (A_fast/A_slow)^a. This ratio is much affected by the presence of o-phenanthroline compared to reaction centers with an empty Q_B pocket or with terbutryn present. Because o-phenanthroline was proposed to hydrogen bind His^L190, whereas terbutryn does not, we suggest that a His^LI90-Fe-His^M217 (the equivalent to His^LI90 in the Q_A pocket) “wire” may be involved in the existence of the two conformational states associated with the two phases of charge recombination. In chromat-ophores from the T₁ (Ser^L223→ Ala; Arg^L217→ His) and T₄ (Tyr^L222→ Phe) mutants no modification of the (A_fast/A_slow)^a ratio is detected, whereas the (A_fast/A_slow)^b ratios are substantially modified compared to the wild type (WT). In the T₃ mutant (Phe^L216→ Ser; Val^M263→ Phe [4.1 Å apart from Q_A]), (A_fast/A_slow)^a is notably changed compared to the WT. Our data show that any modification in the close protein environment of the quinones and/or of the His^L190 and His^M217 affects the equilibrium between the two reaction center states. This is consistent with the existence of two reaction center states from Rps. viridis, associated with two different conformations of the quinones-histidines-iron system. This “wire” allows both quinone protein pockets to interact over quite long distances.     

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b₅ (cytb₅) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb₅ complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb₅ complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.     

Quantitative analysis and evaluation of the solubility of hydrophobic proteins recovered from brain, heart and urine using UV-visible spectrophotometry

There is a need for a simple method that can directly quantify hydrophobic proteins. UV-visible spectrophotometry was applied in the present study for this purpose. Absorbance at λ=280 nm (A ₂₈₀) was detected for both Escherichia coli membrane proteins and bovine serum albumin, whereas absorbance at λ=620 nm (A ₆₂₀) was only detected for E. coli membrane proteins. The A ₆₂₀ values of the brain samples were greater than those of heart samples when equal concentrations were used, regardless of the type of solubilizing agent employed. Because hydrophobic proteins tend to form colloidal microparticles in solution, we also applied UV-visible spectrophotometry to evaluate the efficacies of different extraction protocols for solubilizing hydrophobic proteins. For brain protein extraction, the highest A ₆₂₀ was observed in samples recovered using Tris, whereas the lowest was from samples recovered using SDS. Solubilizing brain tissue with 0.25% SDS (above the CMC) gave a lower A ₆₂₀ than extraction with 0.025% SDS (below the CMC). Addition of 0.25% SDS to samples recovered with Triton caused A ₆₂₀ to drop. A ₆₂₀ could also be used to distinguish between the hydrophobic fractions (pellets) of brain and urine proteins and their hydrophilic fractions (supernatants) prefractionated using high-speed centrifugation. Additionally, an A ₆₂₀/A ₂₈₀ ratio exceeding 0.12 appears to denote highly hydrophobic samples. Our data suggest that direct UV-visible spectrophotometry can be used as a simple method to quantify and evaluate the solubilities of hydrophobic proteins.     

Moderately concentrated solutions of polystyrene. II. Integrated-intensity light scattering as a function of concentration,temperature, and molecular weight

Integrated-intensity light scattering data are reported for moderately concentrated solutions of polystyrene in benzene and in cyclopentane. The benzene system is one for which the second virial coefficient A₂ is large; data obtained over the range 0.5 < A₂Mc < 30, with c the polymer concentration, are analyzed in terms of the (extrapolated) intensity at zero angle and the angular dependence of the intensity. The former is discussed in terms of power law representations based on scaling relations, which are found to represent the data. The latter is discussed in terms of the dependence of the chain dimensions on concentration. With cyclopentane, the behavior is similar for temperatures for which A₂ is near its maximum, but for T near either Θ_U or Θ_L, for which A₂ is zero or small, the angular dependence of the scattering is distinctly different, with the intensity exhibiting a maximum as a function of scattering angle.     

Effect of electrode membrane surface ratio and thickness on lead electrode potential

Summary The lead ion selective electrode (ISE) consisting of PbS-Ag₂S is normally used with a membrane surface of outer layer areaA _o) and inner layer area (A _i) at unity (A _o=A _i). Partial covering of one surface area with an insulating material and keeping the other surface layer intact resulted in different ratios of membrane surface areas exposed to lead solutions. The potential linearly increased with increasing theA _o/A _i ratio and decreased with decreasing theA _o/A _i ratio. The lead ISE potential increased linearly with increasing the membrane thickness, but which required much longer time for a stable potential.

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Wirkung von Oberflächenverhältnis und Dicke der Elektrodenmembran auf das Potential einer Bleielektrode

Zusammenfassung Die aus PbS-Ag₂S bestehende spezifische Bleiionen-Elektrode wird üblicherweise mit einem Oberflächenverhältnis der Außenfläche (A _o) und der Innenfläche (A _i) mitA _o=A _i verwendet. Durch teilweise Abdeckung einer Oberfläche mit isolierendem Material, ohne daß die andere Oberfläche verändert wird, ergaben sich verschiedene Verhältnisse der mit der Bleilösung in Berührung stehenden Oberflächen. Mit dem VerhältnisA _o/A _i stieg das Potential linear an bzw. es fiel mit diesem Verhältnis ab. Das Potential der spezifischen Bleielektrode stieg mit der Dicke der Membran, stabilisierte sich aber wesentlich langsamer.

     

Thermodynamics of plasticized triblock copolymers. I. Theory

The thermodynamic theory of bulk ABA copolymers developed by Leary and Williams is extended to copolymer–solvent systems. Free energy expressions are derived for five hypothetical phase-separated morphologies and evaluated specifically for a polymer with approximately 25% of the A component. The separation temperature, T_s, at which a given morphology will be in equilibrium with a homogeneous mixture, is also evaluated. The major result is the prediction of the T_s(?S) depression, where ?s is the solvent fraction. Depression is maximized when δ_S is equidistant between δ_A and δ_B, but becomes rapidly less when δ_S is outside the δ_A–δ_B range. Morphological favoritism is independent of ?_S and δ_S (model does not apply to preferential precipitation), with a planar microstructure being favored along with microstructures containing domains of B in continuous A for the 25% A polymer.     

Removal of the PsaF Polypeptide Biases Electron Transfer in Favor of the PsaB Branch of Cofactors in Triton X‐100 Photosystem I Complexes from Synechococcus sp. PCC 7002†

Continuous wave (CW) and transient electron paramagnetic resonance studies have implied that when PsaF is removed genetically, the double reduction of A_1A is facile, the lifetime of A_1A^? is shorter and the ratio of fast to slow kinetic phases increases in PS I complexes isolated with Triton X‐100 (Van der Est, A., A. I. Valieva, Y. E. Kandrashkin, G. Shen, D. A. Bryant and J. H. Golbeck [2004] Biochemistry 43 , 1264–1275). Changes in the lifetimes of A_1A^? and A_1B^? are characteristic of mutants involving the quinone binding sites, but changes in the relative amplitudes of A_1A^? and A_1B^? are characteristic of mutants involving the primary electron acceptors, A_0A and A_0B. Here, we measured the fast and slow phases of electron transfer from A_1B^? and A_1A^? to F_X in psaF and psaE psaF null mutants using time‐resolved CW and pump‐probe optical absorption spectroscopy. The lifetime of the fast kinetic phase was found to be unaltered, but the lifetime of the slow kinetic phase was shorter in the psaF null mutant and even more so in the psaE psaF null mutant. Concomitantly, the amplitude of the fast kinetic phase increased by a factor of 1.8 and 2.0 in the psaF and psaE psaF null mutants, respectively, at the expense of the slow kinetic phase. The change in ratio of the fast to slow kinetic phases is explained as either a redirection of electron transfer through A_1B at the expense of A_1A, or a shortening of the lifetime of A_1A^? to become identical to that of A_1B^?. The constant lifetime and the characteristics of the near‐UV spectrum of the fast kinetic phase favor the former explanation. A unified hypothesis is presented of a displacement of the A‐jk(1) α‐helix and switchback loop, which would weaken the H‐bond from Leu₇₂₂ to A_1A, accounting for the acceleration of the slow kinetic phase, as well as weaken the H‐bond from Tyr₆₉₆ to A_0A, accounting for the bias of electron transfer in favor of the PsaB branch of cofactors.     

Pharmacophore generation,atom-based 3D-QSAR,HQSAR and activity cliff analyses of benzothiazine and deazaxanthine derivatives as dual A2A antagonists/MAO‑B inhibitors

Dual inhibition of A_2A and MAO-B is an emerging strategy in neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). In this study, atom-based three-dimensional quantitative structure–activity relationship (3D-QSAR) and hologram quantitative structure–activity relationship (HQSAR) models were generated with benzothiazine and deazaxanthine derivatives. Based on activity against A_2A and MAO-B, two statistically signi?cant 3D-QSAR models (r² = 0.96, q² = 0.76 and r² = 0.91, q² = 0.63) and HQSAR models (r² = 0.93, q² = 0.68 and r² = 0.97, q² = 0.58) were developed. In an activity cliff analysis, structural outliers were identified by calculating the Mahalanobis distance for a pair of compounds with A_2A and MAO-B inhibitory activities. The generated 3D-QSAR and HQSAR models, activity cliff analysis, molecular docking and dynamic studies for dual target protein inhibitors provide key structural scaffolds that serve as building blocks in designing drug-like molecules for neurodegenerative diseases.     

Gymnemasäure,das antisaccharine Prinzip von Gymnema sylvestre R. Br. Isolierungen und Identifizierungen Glykoside und Aglykone 288. Mitteilung

The isolation, characterisation, purity control and probable structure of gymnemic acid, the antisaccharine principle of Gymnema sylvestre R. Br. (Asclepiadaceae) has been reported. According to the results, gymnemic acid is the D-glucuronide of a probably new hexahydroxy-δ¹²-oleanene which has been named gymnemagenin and which is esterified with various combinations of the following five acids: formic, acetic, n-butyric, isovaleric, and tiglic acid. Gymnemic acid is not an absolutely pure substance. It is composed predominantly of gymnemic acid A₁, which is accompanied by components having similar properties, especially the gymnemic acids A₂, A₃ and A₄. The difference between these components probably lies in the absence or presence of one of the five named acids.     

Interfacial behaviour of wheat puroindolines: monolayers of puroindolines at the air–water interface

Puroindolines are among the major basic and cysteine-rich lipid binding proteins of wheat seeds. The interfacial properties of puroindoline-a (PIN-a) and puroindoline-b (PIN-b) are important both from a biological and a technological point of view. In the work reported here, the interfacial characteristics of spread monolayers of wheat puroindolines at the air–water interface were studied at varying subphase compositions using a Langmuir–Blodgett film balance. The compression isotherms (π–A _Sp) were recorded at constant barrier speed (3.3 cm/min). It was observed that both PIN-a and PIN-b form stable monolayers at the air–water interface. The stability of the monolayers was found to be dependent on the subphase composition as well as on the concentration of protein in the spreading solution. When the ionic strength of the subphase is below 0.50, the compression isotherms of both PIN-a and PIN-b remains unaffected with the change in the ionic strength of the subphase; however, when the ionic strength is above 0.50, the compression isotherms of both PIN-a and PIN-b undergo significant change with an increase in the ionic strength of the subphase. A gradual increase in the values of the collapse pressure (π_C) and the limiting area (A ₀) was observed due to an increase in the ionic strength of the subphase from 0.5 to 4.0, which may be correlated with the salt-induced conformational changes of the protein molecule. The presence of NaCl and KCl (ionic strength 1.0) in the subphase has a comparable effect on the compression isotherms of both PIN-a and PIN-b; however, the presence of CaCl₂ (ionic strength 1.0) in the subphase leads to an increase in the values of π_C and A ₀. A change in the pH of the subphase from 3.0 to 7.2 was to have a significant effect on the values of π_C and A ₀, which may be due to the pH-induced alteration of the protein conformation. Received: 8 August 2000 Accepted: 15 December 2000     

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. “CE-SC_XS” methods (where CE-SC_XS is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with “C_X” representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.