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ヨーロッパコマドリ由来クリプトクロム4 (erCRY4)は、青色光吸収時に量子磁気センサーとして機能し、磁気情報を感知する能力「磁覚」に寄与すると考えられているタンパク質である。本発表では、X線小角散乱法によるerCRY4の構造解析研究の一端を紹介する。本解析の結果、erCRY4が青色光(454nm)を吸収すると、C末端付近の構造揺らぎが抑制されるとともに三次構造が全体的に収縮する比較的大きな構造変化を生じ、量子磁気センサーとして機能するための立体構造を形成する可能性が示唆された。, 量子生命科学会第4回大会

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form. The globular clISCA1 protomer assembled to form columnar oligomers, which allowed for the binding of many Fe-S clusters at the interface between clISCA1 protomers. Moreover, the translational diffusion and the columnar oligomerization of clISCA1 were controlled by the external static magnetic field and Fe-S clusters bound to clISCA1. However, the columnar clISCA1 oligomer was not oriented along the external static magnetic field (~1 T) when clCRY4 was not bound to clISCA1. This result indicated that clCRY4 has a function to enhance the magnetic orientational property of clCRY4/clISCA1 oligomer.

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were −3 e Å−2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2–Pro17) revealed that Ala2–Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content ([Asp + Glu] / [Arg + Lys + His] = 9.0) among known TRXs, was chosen to elucidate a catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main chain structure of HsTRX-A is similar to those of homologous TRXs; e.g., root-mean-square deviations for Cα atoms were

一部の磁覚保有種の網膜細胞に存在する蛋白質クリプトクロム (CRY)は、地磁気程度の磁場を検出できる量子磁気センサーとして機能しうることが、多くの研究者によって明らかにされつつある。現在提唱されている仮説では、青色光照射によって生じるCRY内のラジカル対の電子スピンが磁気情報を受容していると考えられている[1,2]。一方、CRYの構造学的・分子論的研究は大幅に不足しており、そのため、量子レベルの微弱な磁気情報をCRYから神経系へ伝達して知覚化する仕組みは明らかにされていない。そこで本研究では、磁気受容機能発揮時（青色光照射下）におけるヨーロッパコマドリ由来CRY4 (erCRY4)の構造学的特徴や分子挙動を、X線小角散乱法（SAXS）及びサイズ排除クロマトグラフィー（SEC）により精査した。放射光施設Photon Factory BL-10Cを利用してSAXS解析を行った結果、erCRY4単量体は、454nmの青色光を照射することにより、最大径Dmaxが77 Åから74 Åへ減少することが明らかになった。また、SAXSデータに基づくerCRY4単量体のAb-initioモデリングを行った結果、青色光照射によりerCRY4の三次構造が収縮することが示唆された。一方、SECによる分析では、青色光照射により、ゲルろ過カラム内のerCRY4の保持時間が短縮されることが明らかになった。これらの結果から、青色光照射は、erCRY4の自己会合性を増大させる構造変化を誘導すると推察された。青色光照射により網膜細胞内のerCRY4の自己会合性が増大すれば、erCRY4が生じるラジカル対の細胞内局所濃度が増大し、これにより、磁気情報のシグナルが増幅される可能性がある。 [1] Ritz T, et al., Biophys J. (2000) 78, 707 [2] Maeda K, et al., PNAS. (2012) 109, 4774, 2021年度量子ビームサイエンスフェスタ

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation have provided new insights into the structure and dynamics of biological molecules inside the cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background scattering from large amounts of cell debris, we successfully extracted structure information for both small globular protein and small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1:60 for protein/crowder and 1:40 for SUV/crowder.

磁覚保有種の網膜には磁気センサー蛋白質クリプトクロム(CRY)が存在する。青色光によってCRY内のFADが励起されると、TrpからFADへ電子が移動してラジカル対を生じる。このラジカル対の電子ペアの電子スピンが磁気情報を検出すると推測される。しかし、CRYの磁気検出反応場や、CRYが受容した情報を神経系に伝達する機構などは未解明である。一方、近年、Fe-Sクラスター輸送蛋白質ISCA1がCRYと複合体化することが明らかになった。その複合体は青色光照射時に弱磁場(~10G)に対して配向する傾向があり、ISCA1はCRYの磁気受容を補助する、もしくは、CRYからの情報伝達を仲介する候補物質の一つに挙げられている。しかし、この説には異論もある。特にISCA1の構造・物性が殆ど未解明であることが大きな課題であった。これまでに我々は、SAXS解析により、カワラバト由来clISCA1が2種類の構造異性体（球状のType-Aと棒状のType-B）を形成することや、Type-Aは4量体以上の柱状の自己会合体を形成し、その会合面に磁化率・磁気異方性を向上するFe-Sクラスターを結合しうることなどを明らかにした。一方、Type-Bの会合は2量体が上限であり、且つ、Fe-Sクラスターを解離することも明らかにした。clISCA1は構造依存的に磁気応答性を発揮する可能性が示された。更に本発表では、SAXS解析によって明らかにした磁場印加時のclISCA1の分子挙動などについて報告する。, 第21回日本蛋白質科学会年会

近年、多くの動物種において、網膜細胞内の蛋白質クリプトクロム(CRY)を量子磁気センサーとして利用する磁覚（磁場の方位や強さを知覚する感覚）の存在が確認されている。青色光によってCRYが含有するフラビン補酵素(FAD)が励起されると、TrpからFADへ電子が移動し、FADとTrpで構成されるラジカル対を生じる。このラジカル対の電子ペアが外部磁場の影響を受けることで一重項状態と三重項状態の項間交差の効率が変化し、その化学反応性の変化が磁覚の要因になると推測されている [1,2]。また、CRYの磁覚への寄与は、CRYを失活したショウジョウバエが磁覚を喪失することなどから実証されている[3]。しかし、CRYが受容した磁気情報を神経系へ伝達する機構は未解明であり、磁気情報伝達を仲介する物質の探索が当該研究分野の大きな課題となっている[4]。磁覚保有種であるカワラバト由来Fe-Sクラスター輸送蛋白質clISCA1は、上記仲介物質の候補の一つである[5]。カワラバト由来clCRY4/clISCA1複合体は、青色光存在下において、地磁気程度の弱磁場(0.4 – 10G)に対しても配向する稀有な性質を有する[5]。我々は、clISCA1の溶液構造・物性を明らかにするためにSAXS解析を行った。その結果、clISCA1は2種類の構造異性体（球状のType-Aと棒状のType-B）を形成し、Type-Aは4量体以上の柱状の自己会合体を形成することが明らかになった。また、SAXSデータと相同蛋白質の結晶構造（PDB：2D2A、1R94、1X0G）に基づいた分子モデリングから、Type-A自己会合体の分子間界面にFe-Sクラスター結合部位が形成されることが示唆された。Type-Aの自己会合は、起磁力・帯磁率向上の要因となり得る多くのFe-Sクラスターの結合を可能にすると考えられる。一方、Type-Bの自己会合は2量体が上限であり、且つ、Fe-Sクラスター結合能を有さないことも明らかになった。Type-AからType-Bへの構造変化は、clISCA1から他の蛋白質へFe-Sクラスターを転移する際に必要な機能である可能性がある。clISCA1は立体構造に応じて異なる機能（磁気受容もしくは伝達機能、Fe-Sクラスター転移機能）を発現する多機能蛋白質である可能性が示された。（JSPS KAKENHI Grant No. JP18K06174） [1] Ritz T, et al., Biophys J. (2000) 78, 707; [2] Maeda K, et al., PNAS. (2012) 109, 4774; [3] Gegear RJ, et al., Nature (2008) 454, 1014; [4] Wu H et al., Sci Rep. (2020) 10, 7364; [5] Qin S, et al., Nat Mater. (2016) 15, 217, 2020年度量⼦ビームサイエンスフェスタ

塩湖や塩田などの高塩濃度環境に生息する極限環境生物・高度好塩菌は外界との浸透圧調整のために細胞内に飽和濃度の塩(～35% K+)を保有する。高度好塩菌が産生する蛋白質（以下、好塩菌蛋白質）はそのような高塩濃度環境においても機能を保持する。好塩菌蛋白質の高塩濃度適合機構は、高含量の酸性アミノ酸による高い可溶性と、低含量の疎水性アミノ酸(Val, Leu, Ile, Phe)による構造の可塑性（塩析の効果によるコア構造の安定化）などの特徴に基づくことが知られる。しかし、それらの特徴は必ずしも普遍的ではない[1]。また、好塩菌蛋白質の高塩濃度適合機構は、例えば有機溶媒中のような低水分活性環境下でも機能する酵素触媒システムなど、新たなバイオテクノロジーの開発に応用できると期待される。これらの背景から、様々な好塩菌蛋白質について更なる構造・機能研究が求められている[1]。 我々は当該研究の一環として、既知の好塩菌蛋白質の中でも特に酸性アミノ酸含量が高い[（Asp + Glu）/（Arg + Lys + His）= 9.0] 高度好塩性古細菌Halobacterium salinarum NRC-1由来チオレドキシン（以下、HsTRX-A）についてX線結晶解析及び円偏光二色性解析により立体構造・溶液構造を明らかにし、高塩濃度適合機構の解明を試みた[2]。PF BL17AおよびSPring-8 BL38B1を利用して決定したHsTRX-Aの立体構造（PDB ID: 6KIL、分解能1.6Å）を相同性のチオレドキシンと比較した結果、HsTRX-Aの活性残基（Cys45およびCys48）の周辺には他のTRXには見られない水和水ネットワークが存在することが明らかになった。この水和水ネットワークは触媒反応に必要なプロトン輸送を効率化し、高塩濃度・低水分活性環境での機能発現に寄与すると推察された。また、HsTRX-Aは長く柔軟なN末端領域（Ala2-Pro17）を有し、この領域が酸性アミノ酸含量の増大や高塩濃度下における構造安定性・可溶性・酵素活性の向上に寄与することが明らかになった。更に、このN末端領域は活性部位を阻害しない位置に伸長しており、HsTRX-Aは高塩濃度適合のために合理的な分子進化を遂げたことが示唆された。 [1] Karan R et al., (2012) Aquat. Biosyst. 8, 4 [2] Arai S et al., (2020) Acta Cryst. D76, 73, 2020年度量子ビームサイエンスフェスタ

Pigeon’s Fe-S cluster binding protein clISCA1 interacts with cryptochrome (clCRY4) which acts as a quantum magnetic sensor. Recently, we revealed that the native structure of clISCA1 protomer is changeable between the globular Type-A and the rod-like Type-B. The Type-A clISCA1 is self-oligomerized toward columnar oligomer, which enables to bind many Fe-S clusters into the interfaces between the clISCA1 molecules in its oligomer and might improve the magnetic sensitivity of clISCA1. Here we report the magnetic effects on the structure and molecular behavior of clISCA1 observed by the small angle X-ray scattering (SAXS) coupled with the periodic Nd-Fe-B permanent magnet circuit. The SAXS data suggested that the oligomerization of clISCA1 proceeds with the applied magnetic field effect. We are planning to confirm whether the magnetic response of clISCA1 observed in this study is a clISCA1-specific phenomenon or not., 量子生命科学会第２回大会

The quantum mechanism for magnetoreception in the biological system has been mainly investigated using cryptochromes (Crys) [Ritz T, et al., Biophys J. (2000) 78, 707; Maeda K, et al., PNAS. (2012) 109, 4774]. However, the signal transduction mechanism from Cry to the nervous system and the brain has not been elucidated yet. Recently, the ISCA1 (also called MagR) protein of pigeon, fruit fly, etc. was found to interact with Cry. The pigeon Cry4/ISCA1 (pCry4/pISCA1) complex orientates along the weak magnetic field (0.4 G) under blue light [Qin S, et al., Nat Mater. (2016) 15, 217], suggesting that ISCA1 is one of the candidates for partners of Cry upon the magnetoreception reaction and/or the signal transduction. In order to elucidate the structure and molecular behavior of pISCA1, we conducted the small angle X-ray scattering (SAXS) analysis of pISCA1. The result indicated that pISCA1 forms two types of conformers; the globular type-A and the rod-like type-B. The type-A conformer was self-polymerized larger than tetramer. Molecular modeling of the pISCA1 based on the SAXS analysis and the crystal structures of homologs (PDB: 2D2A, 1R94 and 1X0G) suggested that Fe-S clusters bind to the interfaces between the type-A pISCA1 molecules in its own oligomer. The conformational change from type-B to type-A and the subsequent self-polymerization of pISCA1 should enable to bind many Fe-S clusters that might improve the magnetomotive force of the pCry4/pISCA1 complex to orientate along the external magnetic field. This work was supported by JSPS KAKENHI Grant Number JP18K06174., 2020WCPS

量子磁気センサーとして機能しうるカワラバト由来蛋白質clCRY4と相互作用する蛋白質clISCA1の機能・物性について、放射光X線小角散乱法と試料磁場印加装置を駆使して得た知見を報告する。また、本研究によって間接的に推察されたclCRY4の機能についても報告する。 clISCA1は、周囲の磁場の状態や、同蛋白質に結合している2Fe-2Sクラスターの酸化還元状態に応じて、柱状の多量体を形成することが明らかになった。また、clCRY4/clISCA1複合体は、青色光照射時に0.4－10G程度の弱磁場に対して配向することが知られるが、clISCA1単独の柱状多量体は1 Tの磁場を印加しても配向しないことが明らかになった。得られた知見から、clCRY4はclCRY4/clISCA1複合体の磁場配向性を増大する未知の機能を有することが示唆された。

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [1] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability. Furthermore, the mutant simultaneously improved a tolerance for protease digestion, e.g. trypsin and proteinase K, and for organic solvent. Residual activity of the mutant after treatment with 10% 2-propanol, 10% DMF and 20% DMSO at 35 °C for 1 h was 29.4, 24.8 and 91.3%, respectively, whereas that of the wild type was 0.4, 0.1 and 24.3%, respectively.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3 M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.

The iron-sulfur cluster assembly 1 homolog clISCA1 of Columba livia (pigeon) interacts with a quantum biosensor protein clCRY4. The clCRY4/clISCA1 complex tends to orientate along the weak external magnetic-field lines (0.4–10G) under blue light, suggesting that clISCA1 might assist the function of clCRY4. It was expected that the Fe-S cluster binging to clISCA1 might improve the magnetic property of clISCA1. In order to elucidate the Fe-S cluster binging mechanism of clISCA1, we conducted the small angle X-ray scattering analysis coupled with size exclusion chromatography analysis (SEC-SAXS) and UV/Vis spectroscopy. The result suggests that the globular protomers of clISCA1 form columnar oligomers, and Fe-S cluster binding sites are formed between clISCA1 protomers in a columnar oligomer. Periodic and regular binding of Fe-S clusters along the long axis of the columnar oligomer may improve the magnetic susceptibility and the magnetic anisotropy of the clISCA1 oligomer and the clCRY4/clISCA1 complex.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 A using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (-1.52 ± 0.05 kJ mol(-1) K(-1) ) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 ∼ 0.25 kJ mol(-1) K(-1) ) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO163 ) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332 ). In order to clarify the structural and physicochemical properties of hTPO163 , circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and -16.7% for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm ) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154-332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3-fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose.

The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr2+-binding sites and one Cs+-binding site were discovered in the HaBLA molecule., Environmentally friendly absorbents are needed for Sr2+ and Cs+, as the removal of the radioactive Sr2+ and Cs+ that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+ or Sr2+. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr2+ and Cs+ ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+ (90 mM Na+/10 mM Cs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+ and Cs+ ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes.

In order to clarify the structural basis of the halophilic characteristics of an alkaline phosphatase derived from the moderate halophile Halomonas sp. 593 (HaAP), the tertiary structure of HaAP was determined to 2.1 Å resolution by X-ray crystallography. The structural properties of surface negative charge and core hydrophobicity were shown to be intermediate between those characteristic of halophiles and non-halophiles, and may explain the unique functional adaptation to a wide range of salt concentrations., Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (Cα r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (-1.52 ± 0.05 kJ mol(-1) K(-1) ) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 ∼ 0.25 kJ mol(-1) K(-1) ) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum , requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S–S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S–S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 A resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 A resolution with space groups of P21 and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

TLS (Translocated in LipoSarcoma), also termed FUS, is a multifunctional protein implicated in diverse cellular events such as maintaining genome integrity and regulating gene expression. We have focused on the role of TLS as a coregulator in transcriptional regulation. In the process of investigating TLS-binding proteins, we found that PRMT1 (protein arginine methyltransferase 1) was in complex with TLS. We analyzed the methylation status of endogenous TLS and demonstrated that TLS was arginine-methylated by PRMT1. Using mass spectrometry, we identified that four arginine residues within TLS (R216, R218, R242 and R394) were consistently dimethylated. We performed luciferase reporter assays to assess the functional consequence of TLS arginine methylation in transcriptional regulation and, interestingly, observed that TLS and PRMT1 synergistically coactivated transcription at the survivin promoter. Further analysis using a catalytic-dead PRMT1 or methylation inhibitor both showed that the synergistic transcriptional activation was mediated by TLS arginine-methylation. These results revealed a cooperative role of TLS and PRMT1 in transcriptional regulation.

The subunit structure of Halomonas nucleoside diphosphate kinase (HaNDK) is a dimer, different from NDKs of other species. We have shown before that it is due to Glu134 in HaNDK, which results in steric hindrance and charge repulsion between two dimeric units and prevents further assembly into the tetramer and that changing the Glu134 to neutral Ala results in formation of a stable tetramer. To our surprise, both wild-type NDK from moderately halophilc Chromohalobacter salexigens (CsNDK/GNE: GNE represents Gly134-Asn135-Glu136) and mutant CsNDK/ A NE, both of which have a neutral amino acid at residue 134, were found to form a dimer. These constructs contain Glu136, which may also cause steric barrier and charge repulsion. A double mutant, CsNDK/ A N T , having Thr at 136 resulted in stable tetrameric assembly, supporting the above notion. A mutant CsNDK/ G N T reverted, however, to a dimer again, indicating that the introduced Ala residue at 134th in the double mutant generated a hydrophobic cluster consisting of the Ala residues and thereby stabilized dimer–dimer association of CsNDK assembly, while Gly destabilized it due to the loss of this cluster. Based on these observations, it is evident that both residues 134 and 136 contribute to the subunit assembly of CsNDK.

With the recent recognition of non-coding RNAs (ncRNAs) flanking many genes, a central issue is to obtain a full understanding of their potential roles in regulated gene transcription programmes, possibly through different mechanisms. Here we show that an RNA-binding protein, TLS (for translocated in liposarcoma), serves as a key transcriptional regulatory sensor of DNA damage signals that, on the basis of its allosteric modulation by RNA, specifically binds to and inhibits CREB-binding protein (CBP) and p300 histone acetyltransferase activities on a repressed gene target, cyclin D1 (CCND1) in human cell lines. Recruitment of TLS to the CCND1 promoter to cause gene-specific repression is directed by single-stranded, low-copy-number ncRNA transcripts tethered to the 5' regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced ncRNAs localized to regulatory regions of transcription units can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of RNA-binding co-regulators in response to specific signals, providing an unexpected ncRNA/RNA-binding protein-based strategy to integrate transcriptional programmes.

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer–dimer interaction and prevent the formation of the tetramer.

We describe here the cloning and characterization of a novel mouse homeodomain-interacting protein kinase (HIPK)-like gene, Hipk4. Hipk4 is expressed in lung and in white adipose tissue and encodes a 616 amino acid protein that includes a serine/threonine kinase domain. We demonstrate that HIPK4 could phosphorylate human p53 protein at serine 9, both in vitro and in vivo. Among known p53-responsive promoters, activity of the human survivin promoter, which is repressed by p53, was decreased by HIPK4 in p53 functional A549 cells. Human BCL2-associated X protein-promoter activity was not affected. These findings suggest that phosphorylation of p53 at serine 9 is important for p53 mediated transcriptional repression.

Nucleoside diphosphate kinase isolated from psychrophilic Pseudoalteromonas sp. AS-131 (ASNDK) was expressed in Escherichia coli and purified to homogeneity. Comparing to mesophilic NDK isolated from Pseudomonas aeruginosa, ASNDK exhibited highly elevated thermolability: E. coli expression at 37 °C as a denatured insoluble form, 30 °C lower optimum temperature of enzymatic activity, and greatly reduced heat stability with 38 °C lower Tm value, fourfold higher Km and reduced Kcat/Km by 0.4-fold upon reaction temperature increase from 20 to 37 °C. The subunit structure of ASNDK was suggested to be dimer, as in NDKs isolated from moderate halophiles.

Amplified in breast cancer 1 (AIB1) is a member of the p160 family of nuclear receptor coactivator protein. Recent studies have reported that high-level AIB1 production is involved in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway for progression to malignant carcinoma in a steroid-independent manner. Here we demonstrate that, in AIB1-knockout DT40 chicken B-lymphocytes, loss of AIB1 results in induction of phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, in addition to the inhibition of DNA replication. In contrast, high-level AIB1 production prevents proapoptotic activation of the JNK/c-Jun signal transduction pathway and induces DNA replication through phosphorylation of the Akt/p65 NF-kappaB subunit RelA under cellular stresses such as UV irradiation or serum deprivation. Moreover, we have found that AIB1 is essential for the phosphorylation of histone H3 at serine 10, which is associated with the signal transduction to chromatin, leading to the transient expression of immediate-early genes in response to UV stimulation. Our results therefore suggest that AIB1 directly links to cell cycle control mechanisms in concern with the balance between apoptosis and proliferation.

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, a technique complimentary to ultra-high-resolution [1, 2] X-ray diffraction. Three different types of neutron diffractometers for biological macromolecules have been constructed in Japan, France and the United States, and they have been used to determine the crystal structures of proteins up to resolution limits of 1.5-2.5 A. Results relating to hydrogen positions and hydration patterns in proteins have been obtained from these studies. Examples include the geometrical details of hydrogen bonds, H/D exchange in proteins and oligonucleotides, the role of hydrogen atoms in enzymatic activity and thermostability, and the dynamical behavior of hydration structures, all of which have been extracted from these structural results and reviewed. Other techniques, such as the growth of large single crystals, the preparation of fully deuterated proteins, the use of cryogenic techniques, and a data base of hydrogen and hydration in proteins, will be described.

By neutron diffraction measurements of B-DNA duplex, we have succeeded in determining the D positions in 27 D2O molecules. Moreover, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using X-ray and neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern, but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG) 2 duplex. Moreover, the D positions in 27 D 2 O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 A resolution X-ray and 3.0 A resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.

The parameters used to evaluate biomacromolecular crystal quality [Rmerge, I/sigma(I), maximum resolution and mosaicity] strongly depend on the experimental diffraction conditions. In this paper, the distinctive features of the relative Wilson plot method are described and it is shown that the overall B factor obtained from this plot is more appropriate for the characterization of protein crystals. The relative Wilson plot has been applied to the characterization of crystals of the B-DNA decamer d(CCATTAATGG) and crystals of the proteins DsrD (dissimilatory sulfite reductase D) and hen egg-white lysozyme (HEWL), which were studied by neutron diffraction. It was found that the crystal quality of the B-DNA decamer and DsrD depended significantly on the regions of the crystallization phase diagram from which the samples were taken. However, in the case of HEWL crystal quality appears to be independent of the region of the crystallization phase diagram.

Differential scanning calorimetry (DSC) measurements are well known to serve us heat capacity functions of macromolecules and molar fractions of thermodynamic microstates. On the other hand, small-angle X-ray scattering (SAXS) measurements are expected to have an advantage for determining directly spatial-conformational states of macromolecules since we can observe ensemble-averaged scattering functions from solute macromolecules at multiple spatial-conformational states. In the present paper we will present an approach to analyze spatial-conformational-state transitions observed in denaturation processes of proteins by SAXS, which affords us a new aspect of thermal transition of proteins in comparison with thermodynamic-microstate transitions observed by DSC. From the point of view of spatial-conformational-state transition, we will clarify the thermal structural transition aspects of hen egg-white lysozyme (HEWL) at pH 5 depending on the conformational hierarchy and concentration.

To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH approximately 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.

In this present paper we present a new method of small-angle X-ray scattering (SAXS) data analysis (called the TMA method) to examine multiplicity of structural transition of denaturation processes of proteins. The TMA method gives us a molar fraction of spatial-conformational-state and an indication of deviation from the two-state structural transition hypothesis in a structural transition process. Using this method we have successfully compared a spatial-conformational-state transition observed by SAXS with a thermodynamicmicrostate transition observed by differential scanning calorimetry (DSC) in the thermal denaturation process of hen egg-white lysozyme (HEWL) at the pH range of 2.4-3.1. The TMA method is able to dissect spatialconformational-state transition multiplicity depending on structural hierarchy. We have found that the thermal structural transition of HEWL is well-characterized mostly in terms of two different spatial-conformationalstate transitions in the tertiary and intramolecular structures. Upon heating, the transition-midpoint temperature of the spatial-conformational-state transition of the tertiary structure mostly agrees with that of DSC, while the transition-midpoint temperature of the intramolecular structure greatly differs from that of DSC. The tertiary structural transition essentially obeys the two-state transition feature with a sharp critical temperature in comparison with the intramolecular structural transition. When the pH is lowered from 3.1 to 2.4, the former transition tends to shows a maximum deviation from the two-state transition feature around the transition temperature. On the other hand, the intramolecular structural transition proceeds continuously with a relatively large deviation in the whole temperature range measured at all pH values. The present results obtained by the TMA method essentially agree with our previous report using standard SAXS analyses in HEWL system at different pH range of 7.0-1.2, and would afford us further aspects to discuss experimental evidences with theoretical models of protein folding, such as nucleation-collapse models.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.

By using small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) we studied the thermal structural transition of lysozyme in the temperature range from 20 to 85 °C at pH 1.2, 3.9, and 7.0. α-Lactalbumin was used as a control. We found that the onset temperature determined from the DSC thermogram mostly coincides with the starting temperature of the evident change of the tertiary structure reflected in the gyration radius and in the distance distribution function obtained by SAXS. The tertiary structural transition of the lysozyme at pH 3.9 and 7.0 occurs sharply as a two-state transition; however, at pH 1.2 this transition proceeds rather gradually. The change of the Porod slope occurred at around the tertiary structural transition temperature and suggests that the elevation of temperature remarkably changes the protein surface from a smooth one to a fractally rough one. On the other hand, the local structural change, namely the intramolecular rearrangement of the polypeptide chain...