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首页> 外文期刊>Biochemistry >Characterization of the lumenal region of human tapasin reveals the presence of two structural domains
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Characterization of the lumenal region of human tapasin reveals the presence of two structural domains

机译：人类塔帕森管腔区域的表征揭示了两个结构域的存在

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摘要

Tapasin is a type I membrane glycoprotein involved with other accessory proteins in the assembly of class I MHC-beta(2)m-peptide complexes in the endoplasmic reticulum. We have probed the three-dimensional structure of the lumenal region of human tapasin (residues 1-392) tagged with a (HiS)(6) sequence at its C-terminus using biochemical and biophysical techniques. The far-UV circular dichroism spectrum revealed that tapasin possesses well-defined secondary structural elements corresponding predominantly to beta-sheets. A thermal denaturation curve recorded at 216 nm showed a midpoint transition centered at similar to45 degreesC. Sedimentation analysis showed that tapasin is monomeric in solution with a sedimentation coefficient, Sdegrees(20,w), of 2.68 S. This value of Sdegrees(20,w), combined with the value of the molar mass obtained by MALDI mass spectrometry (44.2 kDa) yielded a frictional ratio, f/f(0), of 1.47. Assuming tapasin is a prolate ellipsoid, we calculated an apparent length of 22.5 nm and a diameter of 2.62 nm, consistent with an elongated molecular shape. Controlled proteolysis using various enzymes revealed that a narrow region of tapasin near residue 90 is highly susceptible to digestion, resulting in two fragments that are resistant to further cleavage. The identity of these fragments was determined by amino acid sequencing and MALDI mass spectrometry and revealed a 9 kDa N-terminal fragment and a 34 kDa C-terminal fragment. Collectively, these results suggest that tapasin is comprised of two core domains of different sizes loosely linked by a flexible region. [References: 42]
机译：Tapasin是一种I型膜糖蛋白，与内质网中的I类MHC-beta（2）m-肽复合物组装过程中的其他辅助蛋白有关。我们已经使用生化和生物物理技术探测了在其C末端标记有（HiS）（6）序列的人类塔帕森蛋白（残基1-392）的腔区域的三维结构。远紫外圆二色性光谱显示，塔帕森蛋白酶具有定义明确的二级结构元素，主要对应于β-折叠。在216nm处记录的热变性曲线显示了以类似于45℃为中心的中点转变。沉降分析表明，塔帕蛋白酶在溶液中为单体，沉降系数Sdegrees（20，w）为2.68S。该Sdegrees（20，w）的值与MALDI质谱法得到的摩尔质量的值相结合（44.2 kDa）产生的摩擦比f / f（0）为1.47。假设塔帕森蛋白酶是长椭圆形，我们计算出22.5 nm的表观长度和2.62 nm的直径，与细长的分子形状一致。使用各种酶进行的蛋白水解控制表明，靠近残基90的Tapasin的狭窄区域极易消化，从而产生了两个片段，可抵抗进一步切割。通过氨基酸测序和MALDI质谱法确定这些片段的身份，并揭示了9kDa的N末端片段和34kDa的C末端片段。总的来说，这些结果表明，塔帕森蛋白酶由两个不同大小的核心结构域组成，这些结构域通过柔性区域松散地连接。 [参考：42]

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