A Protein Functionalization Platform Based on Selective Modification at Methionine Residues

基于蛋氨酸残基选择性修饰的蛋白质功能化平台

• 批准号: EP/S033912/1
• 负责人: Matthew Gaunt
• 金额: $ 241.18万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FS033912%2F1
• 关键词: Protein; Functionalization; Platform; Based; Selective

Nature routinely carries out site-selective modification of proteins, enabling a dramatic increase in functional diversity. In contrast, synthetic manipulation of proteins is restricted by the availability of suitable chemical transformations. However, access to synthetically modified proteins has become fundamentally important to chemical biology, molecular biology & medicine. This has stimulated intensive research into the development of chemical transformations that are compatible with biological systems. Ideally, a reaction should be selective at a single site on a protein at a rate that is commensurate with the kinetic demands of complex molecules; should operate under ambient conditions to prevent disruption of the protein architecture or function; & provide homogeneous products in near perfect conversion. Despite these challenges, the past 20 years have seen a number of exciting methodologies emerge for executing transformations, both in vitro & in vivo, at natural & non-natural amino-acid residues in proteins. While most chemical methods have focussed on expanding the toolkit for reaction at cysteine (Cys) & lysine (Lys) residues, there has been burgeoning interest in transformations at non-natural amino acids (via genetic encoding) that display side chain functionality with orthogonal reactivity to standard residues. Reagents that probe biological processes tend to rely on relatively simple reactions to circumvent problems with chemistry in complex environments. However, there is a need for complementary tools (to reactions at Cys & Lys) that selectively produce functional protein conjugates via previously unexplored amino acids.Methionine (Met) is a proteogenic amino acids & displays a number of features which make it potentially amenable as a bioconjugation target: For example, Met has a <2% abundance in proteins, is easily encoded, has a limited role in ancillary protein function (mainly protection against oxidative stress) & contains a possible reactive handle via its weakly nucleophilic S-atom. Until recently, practical Met-selective bioconjugation was unknown. Concurrent with our initial work, Chang et al reported a redox-activated tagging strategy that converted Met to sulfoximine-derivatives & successfully applying it in biology-driven applications. We have developed a methionine-selective protein functionalization strategy based on the use of hypervalent iodine reagents (we call these reagents MetSIS, meaning Methionine Selective Iodonium Salts) that react selectively with polypeptides & proteins, often giving >95% conversion to a stable diazo-sulfonium conjugate at low concentration in H2O in <1 minute. Our initial work on Met-bioconjugation was recently published Nature 2018, 562, 563-568.The structural diversity of the proteome in any single organism means that no one protein functionalization method will provide universal solutions to the preparation of protein constructs. Cys-ligation is the benchmark for protein labelling and is is fast, selective and continues to evolve powerful methods that can be applied to a plethora of chemical biology & bio-medical applications. Even though Cys has founded many distinct applications, what if another amino acid can be generally harnessed for protein functionalization that is complementary to Cys? Here, I propose that chemical targeting of methionine (Met) can be the basis of distinct strategies for protein modification. Through this Fellowship, I outline a program for synthesis-driven protein functionalization, where these biomacromolecules are labelled at Met for use in vitro & (possibly) in vivo environments thereby expanding the tools available to chemical biologists. Parallel lines of enquiry (Phases A&B) will generate protein functionalization tools & synergistically feed into a Phase C, which will seek to translate the synthesis advances to application.

自然通常会对蛋白质进行现场选择性修饰，从而使功能多样性的急剧增加。相反，蛋白质的合成操作受合适的化学转化的可用性限制。但是，获得合成修饰的蛋白质的访问已对化学生物学，分子生物学和医学至关重要。这激发了对与生物系统兼容的化学转化发展的深入研究。理想情况下，反应应在蛋白质上的单个位点具有选择性，以与复杂分子的动力学需求相称。应在环境条件下运行以防止蛋白质结构或功能的破坏；并以几乎完美的转换提供同质产品。尽管存在这些挑战，但在过去的20年中，在蛋白质中的天然和非天然氨基酸残基上，出现了许多令人兴奋的方法来执行体外和体内转化。虽然大多数化学方法都集中在扩展半胱氨酸（CYS）和赖氨酸（LYS）残基上反应的工具包，但人们对非自然氨基酸的转化（通过遗传编码）的转化一直引起人们的兴趣，这些转化表现出与对标准残基正交反应性的侧链功能。探测生物过程的试剂倾向于依靠相对简单的反应来规避复杂环境中化学问题的问题。 However, there is a need for complementary tools (to reactions at Cys & Lys) that selectively produce functional protein conjugates via previously unexplored amino acids.Methionine (Met) is a proteogenic amino acids & displays a number of features which make it potentially amenable as a bioconjugation target: For example, Met has a <2% abundance in proteins, is easily encoded, has a limited role in ancillary蛋白质功能（主要是针对氧化应激的保护），并通过其弱亲核的S原子包含一个可能的反应性手柄。直到最近，实用的MET选择性生物缀合尚不清楚。同时，Chang等人报告了一种经过氧化还原激活的标记策略，该策略转换为硫胺衍生物并成功地将其应用于生物学驱动的应用中。我们已经建立了一种基于高价值碘试剂的使用（我们称之为Metsis的试剂，意思是蛋氨酸选择性碘盐），它们与多肽和蛋白质有选择性反应，通常会在低浓度<1分。我们最初关于Met-Bioconjugation的最初工作最近发表了《自然2018》，第562、563-568页。任何单个生物体中蛋白质组的结构多样性都意味着，任何一种蛋白质功能化方法都不会为蛋白质构建体制备提供通用的解决方案。 Cys-Rigation是蛋白质标记的基准，并且是快速，选择性的，并且继续进化了可以应用于多种化学生物学和生物医学应用的强大方法。尽管CYS建立了许多不同的应用，但如果通常可以利用另一种氨基酸来用于互补的CYS蛋白质功能化怎么办？在这里，我建议蛋氨酸（MET）的化学靶向可以是蛋白质修饰的不同策略的基础。通过这项奖学金，我概述了一个用于合成驱动的蛋白质功能化的程序，其中这些生物大分子在Met时标记为在体外使用，并在体内环境中使用，从而扩展了化学生物学家可用的工具。平行询问线（A＆B阶段）将生成蛋白质功能化工具和协同作用为C相C，该工具将寻求转化综合的应用进步。