Structure of a Complete Integrin Ectodomain in a Physiologic Resting State and Activation and Deactivation by Applied Forces

The complete ectodomain of integrin αIIbβ3 reveals a bent, closed, low-affinity conformation, the β knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The β3 knee passes between the β3-PSI and αIIb-knob to bury the lower β leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the β subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state. © 2008 Elsevier Inc. All rights reserved

Pt-Clサ サクタイ ニ オイテ リョウシ コウシユラギ デ ユウキ サレタ パイエルスタン ノ サブギャップ ヒカリ キュウシュウ

The temperature dependence of the low-energy tail of the interband optical absorption band at the Peierls edge has been studied in halogen-bridged linear-chain complexes, [Pt(en)2][Pt(en)2Cl2](ClO4)4 and [Pt(en)2][Pt(en)2Cl2](BF4)4, en being ethylenediamine. The absorption spectrum and its temperature dependence are explained well in terms of the subgap density of states of the one-dimensional electronic bands induced by the quantum vibration of Cl ions of the breathing mode. Both substances are found to have a dimensionless electron-phonon coupling constant of λ≊1.2, the zero-temperature gap parameter of Δ0≊1.45 eV, and the transfer integral of t0≊0.4 eV. Because of the strong electron-phonon coupling the temperature dependence of the energy gap is dominated by the self-energy effect; even at 0 K the self-energy amounts to 0.07–0.08 eV, being considerably greater than the self-energies in usual semiconductors. The role of the anharmonicity of the chainlike Pt-Cl bonds is discussed in relation to the origin of the large gap parameter.パイエルス端から低エネルギー側に裾を引く光吸収帯の温度依存性をClO4塩とBF4塩の2つの擬一次元PtCl錯体について調査した。実験結果は、Ptを架橋しているCl-イオンの量子振動がパイエルス端の低エネルギー側に裾を引くように電子の状態密度を誘起するという理論によってよく説明できることが分かった。実験データより電子・音子結合定数、エネルギーギャップ定数および移動積分が定量的に評価された。これらの値からさらに、擬一次元PtCl錯体特有のセルフエネルギー効果とPt-Cl結合の非調和性にまで踏み込んだ知見が得られた

MXサ サクタイ ニ オケル チョウ ジュミョウ ミッドギャップ ジョウタイ ノ ヒカリ セイチョウ ト ゲンスイ ノ ジカン リキガク

An experimental study on the pumping-power dependence of photogrowth and decay properties of the long-lived midgap absorption band in an MX chain compound [Pt(en)2][Pt(en)2Cl2](BF4)4, (en)=ethylenediamine, is reported. The data show that the upper bound of the density of solitonic photogenerated states is determined by autophotobleaching processes. The observed whole course of the photogrowth and decay can be described well on the basis of a lattice ring model, in which intersite energy barriers serve as the particle source as well as barriers against hopping motion.擬一次元白金錯体[Pt(en)2][Pt(en)2Cl2](BF4)4の結晶中に光で生成される長寿命ミッドギャップ状態の成長と減弱の一連のプロセスを、レーザ照射強度を広い範囲で変えて観察した。得られたデータは、ソリトンが光によって反ソリトンとの対で生成され、拡散による衝突合体および自己光漂白を繰り返して消滅していくとする理論で統一的に説明することができ、これにより、PtCl鎖錯体結晶に光誘起されるソリトンの動力学の全貌が明らかになった

MXサ サクタイ ニ ヒカリ セイセイ サレル チョウ ジュミョウ キンク ノ ショウスイ キコウ

The time evolution of the thermal bleaching of the long-lived, photogenerated midgap absorption band in a PtCl chain compound is examined in a temperature range 220–330 K. The long-lived absorption arises from kinks each confined in a segment of the PtCl chain by energy barriers of the order of 0.5 eV. Because of a random distribution of the barrier height the pair coalescence decay shows a lnt dependence at all temperatures in the experimental range, 0–60 min, of time t after discontinuation of laser pumping.擬一次元白金錯体のBF4塩の結晶に光誘起される長寿命のソリトンが自然消滅をしていく経時変化を220 Kから330 Kまでの範囲の種々の温度で詳細に観察し、ソリトンの運動と相互作用のプロセスを実験的に検討した。これにより、PtCl鎖結合には0.5 eV程度の不均一なエネルギー障壁があり、光誘起されたソリトンはこのエネルギー障壁を跳び越えて酔歩運動をしながら、相互衝突により再結合消滅をしていることが明らかになった。また、このような状況での一次元可動粒子の生存率が、時間をtとして -logt 則に従うという、不規則系の新しい実例であることが確認された

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro

Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR

To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential (<i>E</i>_GSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and <i>E</i>_GSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured <i>in vitro</i> (∼ –300 mV), the intracellular Trx exhibited the redox transition at <i>E</i>_GSH between −250 and −200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of <i>E</i>_GSH and the reduction by Trx reductase and other endogenous molecules

MXサ サクタイ [Pt(en)2] [Pt(en)2Cl2] (BF4)4 ノ ミッドギャップ ジョウタイ ニ タイスル リョウシ ユラギ ノ コウカ

Long-lived midgap states are found to be induced efficiently in a substance of the mixed-valence MX chain complex [Pt(en)2][Pt(en)2Cl2](BF4)4, where en=ethylenediamine, by irradiation with the 488-nm line of an Ar laser polarized perpendicular to the Pt-Cl chain bonds. If the temperature is changed between 10 and 250 K in the dark after laser irradiation, the near-infrared absorption bands due to the photoinduced states show pronounced changes in the spectral position, line shape, and intensity. The behaviors of the mean energy and the second and third moments of the A band at low temperatures can be interpreted well in terms of the quantum fluctuation with an eigenfrequency of the order of 0.01 eV. Above about 100 K, the mean energy and the second and third moments show anomalous temperature dependencies, suggesting that the midgap states walk randomly in shallow and periodic potential wells of the chainlike bonds.PTCl錯体の配位子をこれまでのClO4からBF4に変えた[Pt(en)2][Pt(en)2Cl2](BF4)4では、250 Kでも光誘起ミッドギャップ状態がきわめて安定であることが見出された。 ClO4塩のときと同様に、ミッドギャップ光吸収バンドは顕著な温度変化を示すが、250 K以下の温度でミッドギャップ状態自体はほとんど減弱せず、温度変化は可逆的である。これらの実験結果に配位座標モデルを適用して解析することにより、ソリトンの有効質量、ソリトンに対する周期ポテンシャル、量子揺らぎの固有振動数などが定量的に求められた