Structural and Thermodynamic Studies on Protein G

蛋白G是一些链球菌细胞壁的多结构域成分，通过恒定的Fc区与人免疫球蛋白G(Ig G)的所有亚类结合。链球菌G蛋白的免疫球蛋白结合功能包含在一个由56个氨基酸组成的球状结构域中，命名为GB。本建议的目的是：1)利用核磁共振波谱确定GB及其相关突变体的三级结构；2)以该结构域为模型，利用核磁共振、微量热法、CD、荧光光谱和定点突变等方法研究蛋白质折叠和稳定性；3)用上述方法研究GB与免疫球蛋白重链恒定区(Fc)高亲和力相互作用的分子识别基本机制。长期目标是更好地了解这类Fc受体蛋白的功能，并设计出新的Fc受体蛋白类。%GB是探讨氨基酸序列与结构和稳定性关系的理想蛋白质。它的大小是稳定的独特蛋白质结构的下限。此外，它在没有二硫键或紧密配体结合的情况下实现了它的稳定性。它的体积很小，可以应用许多生物物理技术来研究折叠和稳定性，特别是核磁共振方法。关于GB与Fc结合作用的具体性质或其类别选择性的机制，目前还没有什么信息。由于G蛋白与免疫球蛋白结合具有类特异性，并与免疫球蛋白中定义其类的部分结合，我们相信我们的研究将不仅有助于阐明识别机制，而且还将使设计具有新的类选择性的Fc受体成为可能。具有新特性的Fc受体将在医学和生物技术方面有广泛的应用。

Protein G is a multi-domain component of the cell wall of some Streptococcal species and binds to all subclasses of human immunoglobulin G (IgG) by the constant Fc region. The IgG binding function of Streptococcal Protein G is contained in a globular domain of 56 amino acids designated GB. The objectives of this proposal are 1) to determine the tertiary structure of GB and relevant mutants using NMR spectroscopy; 2) to use this domain as a model to study protein folding and stability using NMR, microcalorimetry, CD, fluorescence spectroscopy and site- directed mutagenesis; and 3) to study the basic mechanisms of molecular recognition involved in the high affinity interaction of GB with the heavy-chain, constant region (Fc) of IgG using the above methods. The long term objective is to better understand the function of this class of Fc receptor proteins and engineer new classes of Fc receptor proteins. %%% GB is an ideal protein to probe the relationship of amino acid sequence to structure and stability. It is on the lower limit in size for a stable unique protein structure. Furthermore it achieves its stability without disulfide bonds or tight ligand binding. Its small size permits the application of numerous biophysical techniques to study the folding and stability, in particular NMR methods. Little information is yet available concerning the specific nature of the binding interaction of GB with Fc or the mechanism of its class selectivity. Because Protein G is class specific in its binding to IgG and binds to the portion of the immunoglobulin that defines its class, we believe that our proposed studies will not only help elucidate the mechanism of recognition but also permit the engineering of Fc receptors with new class selectivities. Fc receptors with new properties will have numerous medical and biotechnological applications.