Biological Regulation Studied In Vitro and In Cellulo with Modified Proteins

项目摘要/摘要 我们开发了一种将23S核糖体RNA结构随机化并选择修饰的技术 核糖体将特定类型的修饰氨基酸结合到蛋白质中，而不是通常通过 野生型核糖体。在体外和体内结合的物种包括核碱基氨基酸， 二肽/二肽模拟物、β-氨基酸、磷酸化氨基酸和糖基化氨基酸。 选定的核糖体能够研究两个关键的生化调节过程，即蛋白质糖基化。 和磷酸化。我们还将修改与核酸相互作用的调节蛋白，使 相互作用的可预测的调节或改变的特定性。我们将举例说明使用蛋白质的新策略 含有不寻常的非蛋白生成氨基酸，其结合需要我们选择的核糖体。 在单个或多个位置产生化学计量的磷酸化蛋白质提供了新的 机遇。这些能力包括验证自然磷酸化并研究其影响的能力。它允许 体内(代谢稳定的)磷酸基团的引入，并使新的策略成为可能 鉴定其磷酸化修饰功能的残基。我们发现IB-Tyr42的磷酸化 不仅放松了对核因子-B的抑制，而且促进了与表达核因子-B的基因的结合率 监管机构。我们计划研究IB-中的另外两个已知磷酸化位点，以及NF-B中的三个已知磷酸化位点，而一些 已知核因子-B中丝氨酸磷酸化的部位，但对于酪氨酸和苏氨酸则不是这样。使用新的战略，我们 已经确定了酪氨酸磷酸化的四个位点，并计划研究新的苏氨酸和丝氨酸磷酸化位点。 大多数哺乳动物蛋白质都是糖基化的，但理解/改变碳水化合物的功能是具有挑战性的。 利用在无细胞系统中选择的核糖体，我们制备了含有GlcNAc-的小鼠干扰素-。 位于29位的天冬氨酸氨基转移酶具有抗病毒活性。该中间受体衬底应该能够 碳水化合物簇转移，产生29位完全糖基化的干扰素-，预计具有抗病毒作用 活动。我们最近将相同的糖基化氨基酸及其过乙酰化类似物引入了一种 模型蛋白在纤维素中的得率很高。这一策略可能会产生与那相同的中间体 在体外制备，但数量要大得多。我们希望用天然蛋白质制备完全糖基化的蛋白质 碳水化合物簇，以简化这些簇，并侧重于包含残基，如唾液酸。 细胞调节通常涉及蛋白质与DNA的相互作用；低纳摩尔亲和力和令人印象深刻的选择性 都是典型的。许多X射线晶体结构可以指导设计变化，但试图改变这些调节 通过改变亲和力或专一性的过程失败了。使用具有碱基氨基酸的Rob蛋白， 我们修改了与它们的MICF DNA配对的结合，实现了更强的结合，并增强了细胞的表型 回应。我们建议进一步研究Rob-MICF DNA的相互作用，以完善我们的设计技术。这个 开发的原理将用于解决锌手指系统中的蛋白质-DNA识别问题。

Project Summary/Abstract We have developed a technique for randomizing 23S ribosomal RNA structure, and for selecting modified ribosomes which incorporate into proteins specific types of modified amino acids not ordinarily incorporated by wild-type ribosomes. Species incorporated both in vitro and in vivo have included nucleobase amino acids, dipeptides/dipeptidomimetics, beta-amino acids, phosphorylated amino acids and glycosylated amino acids. The selected ribosomes enable study of two key biochemical regulatory processes, i.e. protein glycosylation and phosphorylation. We will also modify regulatory proteins that interact with nucleic acids, enabling predictable modulation or altered specificity of interactions. We will exemplify new strategies using proteins containing unusual non-proteinogenic amino acids, whose incorporation requires our selected ribosomes. The creation of proteins phosphorylated stoichiometrically at single or multiple positions affords new opportunities. These include the ability to verify natural phosphorylations, and to study their effects. It permits the introduction of (metabolically stable) phosphate groups in vivo, and has enabled new strategies for identifying residues whose phosphorylation modifies function. We showed that phosphorylation of IB- Tyr42 not only relaxes NF-B inhibition, but facilitates the rate of binding to a gene whose expression NF-B regulates. We plan to study two other known phosphorylation sites in IB-, and three in NF-BWhile some sites of serine phosphorylation in NF-B are known, this is not true for Tyr and Thr. Using a new strategy, we have identified four sites of Tyr phosphorylation, and plan to study new Thr and Ser phosphorylation sites. Most mammalian proteins are glycosylated, but understanding/altering carbohydrate functions is challenging. Using a selected ribosome in a cell free system, we prepared murine interferon- (IFN-) containing GlcNAc- Asn at position 29, which can confer antiviral activity. This intermediate acceptor substrate should enable carbohydrate cluster transfer, producing IFN- fully glycosylated at position 29, and expected to have antiviral activity. We have recently introduced the same glycosylated amino acid, and its peracetylated analogue, into a model protein in very good yield in cellulo. This strategy can potentially produce the same intermediate as that prepared in vitro, but in much larger quantities. We wish to prepare fully glycosylated proteins with natural carbohydrate clusters, to simplify these clusters, and to focus on the inclusion of residues such as sialic acid. Cell regulation often involves protein–DNA interactions; low nanomolar affinities and impressive selectivity are typical. Many X-ray crystal structures could guide design changes, but attempts to alter these regulatory processes by changing affinity or specificity have failed. Using Rob proteins having nucleobase amino acids, we modified binding to their micF DNA partners, realizing stronger binding, and enhanced phenotypic cellular responses. We propose to study the Rob–micF DNA interactions further to refine our design techniques. The principles developed will be used to address protein–DNA recognition in a Zn finger system.