Initialising ...
Initialising ...
Initialising ...
Initialising ...
Initialising ...
Initialising ...
Initialising ...
山口 繁生*; 上久保 裕生*; 栗原 和男; 黒木 良太; 新村 信雄*; 清水 伸隆*; 山崎 洋一*; 片岡 幹雄*
Proceedings of the National Academy of Sciences of the United States of America, 106(2), p.440 - 444, 2009/01
被引用回数：158 パーセンタイル：94.62(Multidisciplinary Sciences)Low barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results as well as on theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is due to the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified 
87% (819/942) of the hydrogen positions in PYP, and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state, the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs, as well as the mechanism of the formation of LBHBs.
山口 繁生*; 上久保 裕生*; 清水 伸隆*; 山崎 洋一*; 今元 泰*; 片岡 幹雄
Photochemistry and Photobiology, 83(2), p.336 - 338, 2007/03
被引用回数：7 パーセンタイル：17.11(Biochemistry & Molecular Biology)中性子結晶構造解析及び高分解能結晶構造解析のために、イェロープロテイン(PYP)の巨大結晶を作成した。巨大結晶は、硫酸アンモニウムを沈殿剤とし、さらに塩化ナトリウムを添加物として加えることにより成長した。得られたサイズは1.5
0.70.7mm
である。この結晶は、0.84
分解能までのX線回折斑点を与えた。構造解析の結果、213個(22.6%)の水素原子を観測することができたが、機能上重要な水素結合に関与する水素などは同定できず、中性子結晶構造解析の必要性が明らかとなった。重水から結晶化した試料を用いた予備的な測定から2.1までの中性子回折像を与えることが示された。
山口 繁生*; 上久保 裕生*; 栗原 和男; 清水 哲哉*; 山崎 洋一*; 黒木 良太; 新村 信雄*; 片岡 幹雄*
no journal, ,
Hydrogen atoms play important roles for many biochemical reactions. In the case of photoactive yellow protein (PYP), protonation/deprotonation reactions on amino acid residues and the following rearrangements in the hydrogen bond network are proposed to be closely related with the conformational changes. To understand the molecular mechanism, it is essential to determine the positions of hydrogen atoms. Neutron crystallographic and high resolution X-ray crystallographic studies are well known to be useful for observation of hydrogen atoms. We could obtain a fairly large and high quality crystal of PYP (2.89 0.85 0.79 mm) which gave X-ray diffraction spots up to 0.7. The high-resolution structural analysis clarified previously unknown hydrogen bonding network related with the tertiary structural change. To confirm the hydrogen positions, we are also carrying out neutron diffraction experiments with the obtained crystal.
山口 繁生*; 上久保 裕生*; 栗原 和男; 黒木 良太; 新村 信雄*; 清水 伸隆*; 山崎 洋一*; 片岡 幹雄*
no journal, ,
Hydrogen bond dominates various physical properties in biological molecules and biological chemical reactions including proton transfer, hydrolysis, and so on. A special type of hydrogen bonds, the short hydrogen bond (SHB), has been proposed to have an important role in protein structure and function. The physical properties of SHBs, however, are still under debate. In the photoactive yellow protein (PYP), a light sensor protein, two SHBs near the chromophore have been revealed by X-ray crystallographic studies. The bond distances of the SHBs are modulated during the photo-reaction, resulting in the local proton transfer. From these facts, it can be postulated that these SHBs play a crucial role in the local proton transfer. Using high-resolution neutron crystallography in combination with high-resolution X-ray crystallography, we identified two deuterium atoms involved in two SHBs in PYP. One SHB, between the chromophore and E46, is identified as a low-barrier hydrogen bond (LBHB); the other, between the chromophore and Y42, is a short ionic hydrogen bond (SIHB). This is the first direct evidence of the coexistence of two distinct SHBs in the ground state of a protein. At the conference, we will present the neutron crystallographic structure and discuss the two SHBs in PYP.
