The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein
The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein
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DOI：
10.1002/prot.21748
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发表时间：
2008-05-01
影响因子：
2.9
通讯作者：
Strambini, Giovanni B.
中科院分区：
文献类型：
--
作者：
D'Auria, Sabato;Staiano, Maria;Strambini, Giovanni B.
大肠杆菌谷氨酰胺结合蛋白(GlnBP)是L谷氨酰胺跨细胞膜主动转运的第一步，大小为26 kDa。GlnBP由两个由两条反平行的β链连接的结构域(称为大和小)组成。大结构域类似于小结构域，但它包含两个额外的α-螺旋和三个更短的反平行的β链。在两个结构域之间形成的深裂隙含有配体结合部位。L与谷氨酰胺的结合导致裂隙闭合，结构发生显著变化，形成所谓的闭合式结构。E蛋白含有两个色氨酸残基(W32和W220)和10个酪氨酸残基。我们使用磷光光谱测量来表征在没有和存在谷氨酰胺的情况下两个色氨酸残基在蛋白质结构中的作用。我们的结果指出，GlnBP的磷光很容易在流体溶液中检测到，其中两个色氨酸残基的发射很容易被磷光寿命的巨大差异区分开来，从而将短寿命指定为W220，将长寿命指定为W32。此外，我们的结果表明，由于Y163近端的分子内猝灭，表面W220的三重态寿命异常短。相反，W32的寿命是几百毫秒长，这意味着周围的多肽有一个有序、紧凑的折叠。对光谱数据进行了分析和讨论，并对GlnBP的三维结构进行了详细检查。
Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport Of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two anti-parallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. e protein contains two tryptophan residues (W32 an W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.