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Pressure and Temperature Effects on Protein Solutions

压力和温度对蛋白质溶液的影响

基本信息

批准号：
08045025
负责人：
SAWAMURA Seiji
金额：
$ 1.28万
依托单位：
Ritsumeikan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08045025/
关键词：
protein pressure Lysozyme

项目摘要

Excess partial molar enthalpy of lysozyme in aqueous alcohol was measured using isothermal titration calorimetry. It was suggested that occupancy by the alcohol of a binding site on the surface of the protein may not be necessary for the alcohol to affect the properties of the protein. Pressure dependences of the solubiity for lysozyme and glycine in water measured. The formaer increased and the latter decreased with increasing pressure though both solubilities increased with increasing temperature. It suggests that the pressrue is one of key parameter to consider the protein hydration in the stand point of the constituent amino acids.High-pressure FT-IR of Myoglobin (Mb) was measured in the range -10-70ﾟC.Pressure dependence of the second structure of Mb follows two state model. The structure irreversibly changes to random coil forming intermolecular beta-sheet with increasing temperature though it becomes reversible at low temperature.It was, succeeded in expressing yeast cytochrome c in E.coli through construction of a plasmid for the co-expression of cytochrome c heme lyase and cytochrome c. The yield of wild-type cytochrome c from this system is relatively high. Variants in which the crucial Met80 ligand to the heme iron is replaced with Ala and that do no support respiration can be expressed in E.coli demonstrated that Lys72 is not trimethyllated postsynthetically as it is when expressed in yeast. This finding is no surprising because presumably E.coli does not posses the transmethylase enzyme that catalyzes this modification reaction.

用等温滴定量热法测定了溶菌酶在乙醇水溶液中的过量偏摩尔焓。有人建议，蛋白质表面上的结合位点的醇占用可能不是醇影响蛋白质性质所必需的。测量了溶菌酶和甘氨酸在水中溶解度的压力依赖性。随着压力的增加，前者的溶解度增加，后者的溶解度降低，但两者的溶解度都随着温度的升高而增加。在-10-70 ℃范围内测定了肌红蛋白（Mb）的高压FT-IR，Mb二级结构的压力依赖性符合二态模型。该结构在低温下可逆，但随着温度的升高不可逆地转变为无规卷曲形成分子间β折叠。通过构建细胞色素c血红素裂解酶和细胞色素c共表达质粒，成功地在大肠杆菌中表达了酵母细胞色素c。该系统野生型细胞色素c的产率相对较高。其中血红素铁的关键Met 80配体被Ala取代并且不支持呼吸的变体可以在大肠杆菌中表达，证明Lys 72不是合成后三甲基化的，因为它在酵母中表达时是这样的。这一发现并不令人惊讶，因为据推测大肠杆菌并不含有催化这种修饰反应的甲基转移酶。