Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide.
Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide.
复制标题
光敏色素 A 的表面形貌源自碘乙酰胺的特定化学修饰。
DOI：
10.1021/bi980834i
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Song,PS
中科院分区：
文献类型：
--
作者：
Lapko,VN;Jiang,XY;Smith,DL;Song,PS
光敏色素是植物光形态建成过程中的光可逆光致变色光开关。光敏色素的分子结构和作用机制尚未完全了解。碘乙酰胺修饰的燕麦光敏色素A（phyA）的胰蛋白酶总消化的完整映射的基础上，通过[14 C]碘乙酰胺的半胱氨酸残基的特异性化学修饰来探测phyA的分子表面形貌。在天然条件下，只有两个半胱氨酸（Cys-158和Cys-311）的N-末端发色团结合结构域的11个半胱氨酸被修改到一个显着的程度。在C-末端结构域中，六个半胱氨酸残基（Cys-715、Cys-774、Cys-809、Cys-869、Cys-961、Cys-995）容易被碘乙酰胺接近。在反应性半胱氨酸残基中，只有半胱氨酸-311显示依赖于光致变色形式（Pr Pfr）的感光体的反应性。令人惊讶的是，在发色团附着位点（Cys-321）附近的Cys-311的修饰对phyA的光谱性质没有任何可检测的影响。N-末端结构域的大多数半胱氨酸（Cys-83、Cys-175、Cys-291、Cys-370、Cys-386、Cys-445、Cys-506）深埋在发色团结合结构域的核心中，因为它们只能在色蛋白变性后被修饰。在C-末端结构域中，仅一个半胱氨酸残基（Cys-939）的修饰需要蛋白质变性。由于所有22个半胱氨酸都可以用碘乙酰胺修饰而不还原色蛋白，因此燕麦phyA不具有任何二硫键。我们发现Cys-311、Cys-774、Cys-961和Cys-995在用于光敏色素分离的条件下可以容易地被部分氧化。燕麦phyA的表面形貌/构象及其在光敏色素介导的信号转导中的蛋白质-蛋白质识别中的作用，讨论了半胱氨酸残基的相对反应性。
Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr ⇆ Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein−protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.