Thornton K, Wang Y, Weiner H, Gorenstein D G
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1993 Sep 15;268(26):19906-14.
Previous NMR studies (Karslake, C., Piotto, M. E., Pak, Y. M., Weiner, H., and Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878) had shown that a 22-amino acid signal peptide of rat liver aldehyde dehydrogenase (ALDH) when bound to a micelle had two amphiphilic alpha-helices, one located at the N terminus and the other at the C terminus. It was shown that deletion of either helix caused the precursor protein not to be imported (Wang, Y., and Weiner, H., (1993) J. Biol. Chem. 268, 4759-4765). The two helices are separated by a Arg-Gly-Pro flexible "linker" region, and to test the role of this linker region in the import and processing of the precursor protein, we deleted it from the ALDH signal peptide and precursor protein. The 19-amino acid signal peptide of ALDH, to which has been added 3 residues at the C terminus and from which has been deleted the 3-residue flexible linker region, has been studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment the peptide contains a single alpha-helical segment that extends almost the entire length of the peptide. NH exchange experiments show residues on the hydrophobic face of the peptide to exchange much more slowly than those of the hydrophilic face. Combined with the previous study, these results suggest that precursor protein import simply requires a sufficiently long amphiphilic helix (or helices) to bind stably to the membrane. The N and C helices of native ALDH are only about 6-8 residues long; this represents only about two turns of a helix, and either helix on its own does not provide enough stabilization to ensure folding and binding to the membrane. The linker-deleted ALDH peptide contains a single helix of 12-14 residues that is long enough to provide a hydrophobic surface that can stably interact with the hydrophobic interior of the membrane. The function of the C helix in the native signal peptide is therefore to enhance the stability and binding of the N-terminal signal to the membrane. Significantly, unlike native ALDH precursor protein, the linker-deleted signal peptide precursor protein could no longer be processed after import into mitochondria. As explained by modeling of the alpha-helix and the NH exchange rate data, the precursor protein requires that the first several residues of the mature protein be part of the hydrophobic membrane associated face of the helix.(ABSTRACT TRUNCATED AT 400 WORDS)
先前的核磁共振研究（Karslake, C., Piotto, M. E., Pak, Y. M., Weiner, H., 和 Gorenstein, D. G. (1990) Biochemistry 29, 9872 - 9878）表明，大鼠肝脏醛脱氢酶（ALDH）的一个22个氨基酸的信号肽与胶束结合时具有两个两亲性α螺旋，一个位于N端，另一个位于C端。研究表明，删除任何一个螺旋都会导致前体蛋白无法导入（Wang, Y., 和 Weiner, H., (1993) J. Biol. Chem. 268, 4759 - 4765）。这两个螺旋由一个精氨酸 - 甘氨酸 - 脯氨酸柔性“连接子”区域隔开，为了测试该连接子区域在前体蛋白导入和加工中的作用，我们从ALDH信号肽和前体蛋白中删除了它。对ALDH的19个氨基酸的信号肽进行了研究，该信号肽在C端添加了3个残基，并删除了3个残基的柔性连接子区域，通过二维核磁共振在十二烷基磷酸胆碱胶束中进行。在这种类似膜的环境中，该肽包含一个几乎延伸到肽全长的单一α螺旋片段。NH交换实验表明，肽疏水面上的残基交换速度比亲水面上的残基慢得多。结合先前的研究，这些结果表明前体蛋白的导入仅仅需要一个足够长的两亲性螺旋（或多个螺旋）以稳定地结合到膜上。天然ALDH的N螺旋和C螺旋仅约6 - 8个残基长；这仅代表螺旋的大约两圈，单独的任何一个螺旋都不能提供足够的稳定性来确保折叠并结合到膜上。删除连接子的ALDH肽包含一个12 - 14个残基的单一螺旋，其长度足以提供一个能够与膜的疏水内部稳定相互作用的疏水表面。因此，天然信号肽中C螺旋的功能是增强N端信号与膜的稳定性和结合。值得注意的是，与天然ALDH前体蛋白不同，删除连接子的信号肽前体蛋白导入线粒体后不再能够被加工。正如通过α螺旋建模和NH交换速率数据所解释的，前体蛋白要求成熟蛋白的前几个残基是螺旋疏水膜相关面的一部分。（摘要截选至400字）
