Boutillon C, Wintjens R, Lippens G, Drobecq H, Tartar A
Chimie des Biomolecules, Centre National de la Recherche Scientifique Unité 1309, Faculté de Pharmacie, Lille, France.
Eur J Biochem. 1995 Jul 1;231(1):166-80.
The 55-amino-acid B1-domain of the streptococcal protein G shows a high binding affinity to IgG isolated from a wide range of mammalian species. Since the B1-domain forms an extremely stable globular folding unit containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for protein folding and stability studies. Its small size makes this protein an ideal candidate for production by chemical synthesis, allowing incorporation of non-natural amino acids with the possibility of assessing the influence of such residues on both the functional and structural characteristics of proteins. In this study, we employed three successive chemical syntheses of the B1-domain in order to define the optimal conditions of coupling and protection. The stepwise solid-phase methodology using the tertbutyloxycarbonyl/benzyl strategy was used for this purpose. First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optimized synthesis was performed leading to formation of a synthetic B1-domain with a higher yield; the synthetic B1-domain was completely functional in its binding properties to IgG. Three orthogonal purification steps (gel-permeation, reverse-phase and ion-exchange HPLC) were required to obtain a sample suitable for structural analysis by high-resolution NMR. This study led to the conclusion that the synthetic B1-domain adopts a three-dimensional structure identical to that of the molecule obtained by recombinant techniques [Gronenborn, A.M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) Science 253, 657-661]. To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen alpha-15N-labelled amino acids were incorporated at selected critical positions during the third synthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1-domain.
链球菌蛋白G的55个氨基酸的B1结构域对从多种哺乳动物物种中分离出的IgG具有高结合亲和力。由于B1结构域形成了一个极其稳定的球状折叠单元，包含主要的二级结构元件，且不含脯氨酸残基和二硫键，因此它也是蛋白质折叠和稳定性研究的有用工具。其小尺寸使该蛋白成为化学合成生产的理想候选物，允许掺入非天然氨基酸，从而有可能评估此类残基对蛋白质功能和结构特征的影响。在本研究中，我们对B1结构域进行了三次连续的化学合成，以确定偶联和保护的最佳条件。为此采用了使用叔丁氧羰基/苄基策略的逐步固相方法。首先，评估了序列组装的困难。在分析组装过程中发现的问题后，进行了第二次优化合成，得到了产率更高的合成B1结构域；合成的B1结构域在与IgG的结合特性方面完全具有功能。需要三个正交纯化步骤（凝胶渗透、反相和离子交换HPLC）来获得适合通过高分辨率NMR进行结构分析的样品。本研究得出结论，合成的B1结构域采用的三维结构与通过重组技术获得的分子相同[格罗嫩伯恩，A.M.，菲尔普拉，D.R.，埃西格，N.Z.，阿查里，A.，惠特洛，M.，温菲尔德，P.T.和克洛雷，G.M.（1991年）《科学》253，657 - 661]。为了证明化学方法在一级结构中特异性引入标记氨基酸的有用性，在第三次合成过程中，在选定的关键位置掺入了14个α-15N标记的氨基酸。该类似物是计划详细研究B1结构域折叠动力学的一系列分子中的第一个。
