Biological Regulation Studied In Vitro and In Cellulo with Modified Proteins

项目总结/摘要 我们开发了一种随机化23 S核糖体RNA结构的技术， 核糖体将通常不被核糖体掺入的特定类型的修饰氨基酸掺入蛋白质中， 野生型核糖体。在体外和体内掺入的物质包括核碱基氨基酸， 二肽/二肽模拟物、β-氨基酸、磷酸化氨基酸和糖基化氨基酸。 选择的核糖体使得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， 可预测的调制或改变的相互作用的特异性。我们将研究新的策略， 含有不寻常的非蛋白质氨基酸，其掺入需要我们选择的核糖体。 在单个或多个位置化学计量地磷酸化的蛋白质的产生提供了新的 机会这些包括验证天然磷酸化作用和研究其影响的能力。它允许 在体内引入（代谢稳定的）磷酸基团，并使新的策略， 鉴定其磷酸化修饰功能的残基。我们发现I型B-酪氨酸42的磷酸化 不仅放松NF-κ B B抑制，而且促进与表达NF-κ B B的基因结合的速率， 规范。我们计划研究另外两个已知的I-B-磷酸化位点和三个NF-κ B B磷酸化位点。 NF-κ B B中的丝氨酸磷酸化位点是已知的，但Tyr和Thr的情况并非如此。采用新的策略，我们 已经鉴定了Tyr磷酸化的四个位点，并计划研究新的Thr和Ser磷酸化位点。 大多数哺乳动物蛋白质是糖基化的，但理解/改变碳水化合物功能是具有挑战性的。 在无细胞系统中使用选择的核糖体，我们制备了含有GlcNAc-的小鼠干扰素-γ（IFN-γ）。 29位的Asn，其可赋予抗病毒活性。这种中间受体底物应该能够 糖簇转移，产生IFN-γ，在29位完全糖基化，并预期具有抗病毒活性。 活动我们最近将相同的糖基化氨基酸及其全乙酰化类似物引入了一个 模型蛋白在纤维素中的产率非常高。这种策略可能会产生相同的中间体 在体外制备，但数量要大得多。我们希望制备具有天然糖基化的完全糖基化的蛋白质， 碳水化合物簇，以简化这些簇，并专注于包括残基，如唾液酸。 细胞调节通常涉及蛋白质-DNA相互作用;低纳摩尔亲和力和令人印象深刻的选择性 是典型的。许多X射线晶体结构可以指导设计的变化，但试图改变这些监管 通过改变亲和性或特异性的过程失败了。使用具有核碱基氨基酸的Rob蛋白， 我们修改了与其micF DNA伴侣的结合，实现了更强的结合，并增强了表型细胞 应答我们建议进一步研究Rob-micF DNA相互作用，以完善我们的设计技术。的 开发的原理将用于解决锌指系统中的蛋白质-DNA识别。

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.