A characterization of the lumenal region of human tapasin reveals the presence of two structural domains.
A characterization of the lumenal region of human tapasin reveals the presence of two structural domains.
复制标题
人塔帕蛋白管腔区域的表征揭示了两个结构域的存在。
DOI：
10.1021/bi020521u
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Bouvier,Marlene
中科院分区：
文献类型：
--
作者：
Chen,Mingnan;Stafford,WalterF;Diedrich,Gundo;Khan,Amir;Bouvier,Marlene
Tapasin是一种I型膜糖蛋白，与其他辅助蛋白参与内质网I类MHC - β2m -肽复合物的组装。我们利用生物化学和生物物理技术探测了在c端标记有（His）6序列的人tapasin（残基1 - 392）的管腔区域的三维结构。远紫外圆二色光谱显示，tapasin具有明确的二级结构元素，主要与β-片相对应。在216 nm处记录的热变性曲线显示了以~ 45℃为中心的中点转变。沉淀分析表明，tapasin在溶液中为单体，沉淀系数S°20，w为2.68 S，该S°20值与MALDI质谱法获得的摩尔质量（44.2 kDa）相结合，得到的摩擦比f/f0为1.47。假设tapasin是一个长形椭球体，我们计算出表观长度为22.5 nm，直径为2.62 nm，与拉长的分子形状一致。利用各种酶进行的受控蛋白水解表明，在残基90附近的tapasin狭窄区域极易被消化，导致两个片段无法进一步切割。通过氨基酸测序和MALDI质谱分析确定了这些片段的身份，发现一个9 kDa的n端片段和一个34 kDa的c端片段。总的来说，这些结果表明tapasin由两个不同大小的核心结构域组成，由一个灵活的区域松散连接。
Tapasin is a type I membrane glycoprotein involved with other accessory proteins in the assembly of class I MHC−β2m−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)6sequence 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 β-sheets. A thermal denaturation curve recorded at 216 nm showed a midpoint transition centered at ∼45 °C. Sedimentation analysis showed that tapasin is monomeric in solution with a sedimentation coefficient,S°20,w, of 2.68 S. This value ofS°20,wcombined with the value of the molar mass obtained by MALDI mass spectrometry (44.2 kDa) yielded a frictional ratio,f/f0, 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.