Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus
Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus
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DOI：
10.1021/bi9628883
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Montfort, WR
中科院分区：
文献类型：
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作者：
Andersen, JF;Champagne, DE;Montfort, WR
来自吸血蝽（Rhodnius prolixus）唾液腺的一种一氧化氮转运蛋白（nitrophorin I）已在大肠杆菌中以不溶形式表达，并用血红素进行重组，对其一氧化氮结合动力学和平衡进行了表征。重组nitrophorin I的一氧化氮结合和吸收光谱与来自昆虫的蛋白无法区分。nitrophorin I的一氧化氮结合程度、释放速率以及索雷特吸收最大值均依赖于pH。在pH 5.0 - 8.3范围内，一氧化氮解离常数增加了9倍，从0.19×10⁻⁶增加到1.71×10⁻⁶。在pH 5.0和pH 8.3之间，一氧化氮解离速率增加了2500倍，从1.2×10⁻³增加到3.0 s⁻¹。因此，一氧化氮结合速率也必然依赖于pH，并且在pH 5.0时降低了约280倍。这些因素与nitrophorin的功能相符：在昆虫唾液腺明显的低pH环境中储存一氧化氮，并将一氧化氮释放到昆虫宿主的组织中，从而诱导血管舒张。讨论了一氧化氮结合的可逆性（这在大多数其他血红素蛋白中不会发生）以及对一氧化氮释放的明显动力学控制。我们还报道了适用于通过X射线晶体学确定结构的nitrophorin I晶体。最有希望的晶型在不对称单元中包含两个蛋白质分子，并且衍射分辨率超过2.0埃。
A nitric Oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by similar to 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 Angstrom resolution.