Expanding the biochemical toolbox for protein modification at cysteine

扩展半胱氨酸蛋白质修饰的生化工具箱

• 批准号: EP/R008973/1
• 负责人: Derek MacMillan
• 金额: $ 47.82万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FR008973%2F1
• 关键词: Expanding; biochemical; toolbox; protein; modification

For decades the superior nucleophilicity of the cysteine thiol, relative to other functionality present in proteinogenic amino acids, has been recognised and exploited to enable selective protein modification. However, there are still several untapped opportunities for cysteine-directed biological investigations as a consequence of the difficulty in distinguishing between free cysteine residues of similar availability, particularly in an expressed and/or unfolded polypeptide chain. In order to take full advantage of the targeting potential that cysteine can provide, it is essential that the various cysteine residues within a peptide sequence can be distinguished from each other. Due to the structural similarity between N-epsilon-acetyllysine and 2-acetamidomethyl cysteine (Cys(Acm)) we aim to develop the orthogonal t-RNA/RNA synthetase pair from Methanosarcina barkeri (Mb), employed to incorporate N-epsilon-acetyllysine into expressed proteins in response to an amber (TAG) stop codon, to introduce Cys(Acm). Cys(Acm) is compatible with most routine peptide transformations, importantly with metal free dethiylation (MFD), yet is cleaved under mild conditions, enabling discrimination between available cysteine residues regardless of their sequence context. This protection strategy is currently only available in synthetic peptide chemistry, yet here we aim to use fully recombinant precursors that can be generated cost-effectively from renewable resources.Initially we will randomise Mb tRNA synthetase residues that are proximal to the substrate giving rise to a library of mutant enzymes. Those which can successfully charge Mb tRNA with Cys(Acm) will be amplified and isolated using an established genetic selection. While this progresses we additionally aim to synthesise our protein targets, albeit in simplified form, in order to optimise "late-stage" modification and peptide/protein refolding. Rather than serving as a needless duplication of effort, the synthetic work is designed to create new opportunities for innovation in the protein ligation arena. First, the protection/targeting strategy will first be applied to a model therapeutic protein. Expression of this protein with 2 x Cys(Acm) and a single glycosylation site replaced with cysteine allows unambiguous targeting of the glycosylation site with sugar bromoacetamides. Following glycosylation and deprotection of the Acm groups the protein will be oxidatively refolded. Next the process will be applied to designed tetratricopeptide repeats (TPR's). Head-to-tail oligomerisation of these designed repeat proteins has been shown produce a diverse range of protein-protein interaction scaffolds. However the existing strategy is limited by repeated exposure of the growing polypeptide to proteases, and the introduction of one new cysteine residue per native chemical ligation (NCL) step which results in unwanted disulphide bond formation. Following introduction a single N-terminal Acm protected cysteine residue, iterative NCL/MFD reactions obviates the need for repeated exposure to proteases and prevents interference from free cysteine residues as the oligomerisation progresses. Finally, protected Cys residues will be introduced to a single chain insulin analogue. Whether Insulin is produced chemically or biologically the major factor compromising efficiency is the oxidation step to form the disulfide bonds. Consequently, in order to bias formation of a crucial folding intermediate one pair of cysteine residues (corresponding to A7-B7) will be expressed in Acm protected form. Folding will be examined using standard biophysical techniques including NMR and CD spectroscopy, and compared with a synthetic counterpart. Successful completion of each model study serves as an important proof of principle and highlights the versatility, and wealth of potential applications for this new addition to the protein toolkit.

几十年来，半胱氨酸硫醇相对于蛋白质氨基酸中存在的其他功能性的上级亲核性已被认识到并被利用以实现选择性蛋白质修饰。然而，由于难以区分具有相似可用性的游离半胱氨酸残基，特别是在表达的和/或未折叠的多肽链中的游离半胱氨酸残基，仍有几个未开发的机会用于半胱氨酸定向的生物学研究。为了充分利用半胱氨酸可以提供的靶向潜力，肽序列内的各种半胱氨酸残基可以彼此区分是必要的。由于N-α-乙酰赖氨酸和2-乙酰氨基甲基半胱氨酸（Cys（Acm））之间的结构相似性，我们的目标是开发来自巴氏甲烷八叠球菌（Mb）的正交t-RNA/RNA合成酶对，用于响应琥珀（TAG）终止密码子将N-α-乙酰赖氨酸掺入表达的蛋白质中，以引入Cys（Acm）。Cys（Acm）与大多数常规肽转化相容，重要的是与无金属脱硫基化（MFD）相容，但在温和条件下裂解，使得能够区分可用的半胱氨酸残基，而不管其序列背景如何。这种保护策略目前仅在合成肽化学中可用，但在这里，我们的目标是使用完全重组的前体，它可以从可再生资源中经济有效地产生。最初，我们将随机化邻近底物的Mb tRNA合成酶残基，从而产生突变酶库。将使用已建立的遗传选择扩增和分离能够成功地用Cys（Acm）装载Mb tRNA的那些。在这一进展的同时，我们还旨在合成我们的蛋白质靶点，尽管是以简化的形式，以优化“后期”修饰和肽/蛋白质重折叠。而不是作为一个不必要的重复努力，合成工作的目的是创造新的机会，在蛋白质连接竞技场的创新。首先，保护/靶向策略将首先应用于模型治疗性蛋白质。用2 x Cys（Acm）和用半胱氨酸替换的单个糖基化位点表达该蛋白质允许用糖溴乙酰胺明确靶向糖基化位点。在糖基化和Acm基团的脱保护之后，蛋白质将被氧化重折叠。接下来，该过程将应用于设计的三肽重复序列（TPR）。这些设计的重复蛋白质的头-尾寡聚化已经显示产生多种蛋白质-蛋白质相互作用支架。然而，现有的策略受限于生长的多肽重复暴露于蛋白酶，以及每个天然化学连接（NCL）步骤引入一个新的半胱氨酸残基，这导致不需要的二硫键形成。在引入单个N-末端Acm保护的半胱氨酸残基后，迭代NCL/MFD反应避免了重复暴露于蛋白酶的需要，并防止随着寡聚化的进行而受到游离半胱氨酸残基的干扰。最后，将保护的Cys残基引入单链胰岛素类似物。无论胰岛素是化学还是生物学产生的，影响效率的主要因素是形成二硫键的氧化步骤。因此，为了偏向关键折叠中间体的形成，一对半胱氨酸残基（对应于A7-B7）将以Acm保护的形式表达。折叠将检查使用标准的生物物理技术，包括NMR和CD光谱，并与合成的对应物进行比较。每个模型研究的成功完成都是重要的原理证明，并突出了这种蛋白质工具包新添加的多功能性和丰富的潜在应用。

项目成果

Investigation of acyl transfer auxiliary-assisted glycoconjugation for glycoprotein semi-synthesis.

用于糖蛋白半合成的酰基转移辅助糖缀合的研究。

• DOI: 10.1039/d2ob01633h
• 发表时间: 2022
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Nyandoro K