Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange
Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange
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DOI：
10.1074/jbc.m608417200
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发表时间：
2007-02-23
影响因子：
4.8
通讯作者：
Goto, Yuji
中科院分区：
文献类型：
--
作者：
Lee, Young-Ho;Tamura, Kosuke;Goto, Yuji
核磁共振检测酰胺质子氢/氘（H/D）交换是研究溶液中基于残基的蛋白质构象稳定性和动力学的有力方法。玉米铁氧化还蛋白-NADP（+）还原酶（FNR）是一种较大的蛋白，含有314个氨基酸残基，由黄素腺嘌呤二核苷酸（FAD）和烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）结合域组成。为了研究FNR的结构稳定性和动力学特性，在pD值为8.0和6.0时，利用异核磁共振模拟叶绿体内部的生理相关条件，进行了酰胺质子的H/D交换。在两种pD值下，根据残留物的不同，汇率变化很大。保护残基的谱图表明，高度保护区域与晶体结构提示的疏水核匹配良好，NADP（+）结合域可分为两个亚域。通过核磁共振H/D交换得到的FNR的整体稳定性高于化学变性，这表明H/D交换对于分析基于残基的大蛋白质构象稳定性特别有用，因为大蛋白质的整体展开大多是不可逆的。有趣的是，在白天pH值为pD， 8.0时，叶绿体中NADP（+）结合域的c端亚域的动态构象比pD， 6.0时更可能参与NADP（+）结合的增加，从而提高FNR的活性。在光合作用方面，本研究首次为溶液中fnr型家族提供了基于结构的动力学与功能关系。
NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD, values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD, values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD, 8.0, the daytime pH in chloroplasts, than at pD, 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.