The use of variable density self-assembled monolayers to probe the structure of a target molecule
The use of variable density self-assembled monolayers to probe the structure of a target molecule
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DOI：
10.1016/s0006-3495(98)77640-4
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发表时间：
1998-10-01
影响因子：
3.4
通讯作者：
Bamdad, C
中科院分区：
文献类型：
--
作者：
Bamdad, C
VP16是单纯疱疹病毒编码的一种蛋白，具有78个氨基酸的酸性激活结构域。当与DNA相连时，取自该区域的八个氨基酸基序的串联重复刺激附近基因的转录。这项工作研究了这些最小的激活基序如何与一个假定的靶标--通用转录因子TATA盒结合蛋白(TBP)相互作用，以及这一作用机制的生物学相关性。我开发了新的生物物理技术来区分三种可能的机制模型，这些模型描述了重复的肽基序如何协同影响转录：1)肽基序同时与TBP上的准相同位点结合，产生高亲和力的二价相互作用，从而在转录起始位置附近保持一般的转录因子；2)一个识别基序的结合导致变构效应，从而增强随后额外的肽基序的结合；或者3)肽重复序列与TBP之间确实发生了高亲和力的相互作用，但它不是“二价”相互作用的结果，而是目标蛋白与全长多肽之间多个相互作用的总和。我生成了自组装单分子层(SAM)，它们在二维阵列中呈现不同密度的激活基序多肽，以测试亲和力效应。用表面等离子共振(SPR)来测量靶标(TBP)结合量与多肽密度的函数关系；发现亲和力显著增加，超过一个特征的临界多肽表面密度。竞争抑制实验比较了重复两次或四次的多肽基序和柔性连接子分离的单一基序的亲和力。与TBP预先孵育的四次迭代，抑制了其与类似于高密度多肽表面的结合--比两次迭代好250倍。由柔性氨基酸连接子连接的单肽基序几乎能抑制TBP与表面多肽的结合，并抑制4个串联重复序列。结果支持机制模型1：重复的激活基序与TBP通过高亲和力相互作用，这是单个基序同时结合到TBP上不同位点的协同作用的结果。这一发现与DNA结合的激活结构域通过将通用转录因子TBP拴在转录起始位置附近来触发附近基因转录的想法是一致的。
VP16, a protein encoded by herpes simplex virus, has a well-characterized 78 amino acid acidic activation domain. When tethered to DNA, tandem repeats of an eight amino acid motif taken from this region stimulate the transcription of a nearby gene. This work addresses how these minimal activation motifs interact with a putative target, the general transcription factor TATA box binding protein (TBP), and the biological relevance of this mechanism of action. I developed novel biophysical techniques to discriminate among three possible mechanistic models that describe how reiterated peptide motifs could synergistically effect transcription: 1) the peptide motifs simultaneously bind to quasi-identical sites on TBP, producing a high-affinity bivalent interaction that holds the general transcription factor near the start site of transcription; 2) the binding of one recognition motif causes an allosteric effect that enhances the subsequent binding of additional peptide motifs; or 3) a high-affinity interaction between the peptide repeats and TBP does occur, but rather than being the result of a "bivalent" interaction, it results from the summation of multiple interactions between the target protein and the entire length of the peptide. I generated self-assembled monolayers (SAMs) that presented different densities of the activation motif peptide in a two-dimensional array to test for avidity effects. Surface plasmon resonance (SPR) was used to measure the amount of target (TBP) binding as a function of the peptide density; a marked increase in avidity above a characteristic, critical peptide surface density was found. Competitive inhibition experiments were performed to compare the avidity of peptide motifs, tandemly repeated two or four times, and single motifs separated by a flexible linker. Four iterations of the motif, preincubated with TBP, inhibited its binding to high-density peptide surfaces similar to 250-fold better than two iterations. Single peptide motifs joined by a flexible amino acid linker inhibited TBP binding to surface peptide nearly as well as four tandem repeats. The results favor mechanistic model 1: reiterated activation motifs interact with TBP through a high-affinity interaction that is the result of the cooperative effect of single motifs simultaneously binding to separate sites on TBP. This finding is consistent with the idea that DNA-bound activation domains trigger the transcription of a nearby gene by tethering the general transcription factor, TBP, near the start site of transcription.