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.

大自然通常会对蛋白质进行位点选择性修饰，从而使功能多样性大幅增加。相反，蛋白质的合成操作受到合适的化学转化的限制。然而，获得合成修饰的蛋白质对化学生物学、分子生物学和医学已经变得至关重要。这刺激了深入研究与生物系统相容的化学转化的发展。理想情况下，反应应该在蛋白质上的单个位点以与复杂分子的动力学需求相称的速率进行选择性;应该在环境条件下操作以防止蛋白质结构或功能的破坏;并以近乎完美的转化提供均质产物。尽管存在这些挑战，但在过去的20年里，已经出现了许多令人兴奋的方法，用于在体外和体内对蛋白质中的天然和非天然氨基酸残基进行转化。虽然大多数化学方法都集中在扩大半胱氨酸（Cys）和赖氨酸（Lys）残基反应的工具包，但人们对非天然氨基酸（通过遗传编码）的转化产生了越来越大的兴趣，这些氨基酸显示出与标准残基具有正交反应性的侧链功能。探测生物过程的试剂往往依赖于相对简单的反应，以规避复杂环境中的化学问题。然而，需要有补充工具甲硫氨酸（Met）是一种蛋白质生成氨基酸，并显示出许多特征，这些特征使其可能适合作为生物缀合靶标：例如，Met在蛋白质中具有<2%的丰度，易于编码，在辅助蛋白质功能中的作用有限（主要是保护免受氧化应激），并通过其弱亲核S原子含有可能的反应手柄。直到最近，实用的Met选择性生物缀合是未知的。与我们最初的工作同时，Chang等人报道了一种氧化还原激活的标记策略，将Met转化为亚砜亚胺衍生物，并成功地将其应用于生物驱动的应用中。我们已经开发了一种基于使用高价碘试剂（我们称这些试剂为MetSIS，意思是甲硫氨酸选择性碘鎓盐）的甲硫氨酸选择性蛋白质官能化策略，其选择性地与多肽和蛋白质反应，通常在<1分钟内在低浓度的H2O中将>95%转化为稳定的重氮锍缀合物。我们关于Met-bioconjugation的初步工作最近发表在Nature 2018，562，563- 568中。任何单一生物体中蛋白质组的结构多样性意味着没有一种蛋白质功能化方法可以为蛋白质构建体的制备提供通用的解决方案。半胱氨酸连接是蛋白质标记的基准，具有快速，选择性和持续发展的功能强大的方法，可应用于大量的化学生物学和生物医学应用。尽管半胱氨酸已经有了许多独特的应用，但如果另一种氨基酸可以被用于蛋白质功能化，而这种氨基酸与半胱氨酸互补，那会怎么样呢？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，我概述了一个合成驱动的蛋白质功能化的计划，其中这些生物大分子在Met标记，用于体外和（可能）体内环境，从而扩大了化学生物学家可用的工具。平行的研究路线（阶段A和B）将产生蛋白质功能化工具，并协同进入阶段C，这将寻求将合成进展转化为应用。