Untersuchungen zur Auswirkung von Protein-Glycosylierungen auf das Faltungsverhalten von Proteinen

最近，肽合成与表达蛋白连接的结合已被确立为获得足够数量的纯蛋白的强大工具，用于各种生物物理和医学研究。这种化学和生化方法的结合，其中较小的合成肽部分通过天然化学连接连接到较大的表达蛋白片段，特别有吸引力，因为它允许在合成肽部分进行各种修饰，从而导致以前无法获得的蛋白质的新变体。这种修饰可以包括掺入非天然氨基酸或构象受限的构建块，附着用于监测活性的特定标签或简单地改变蛋白质序列中的整个结构域。在我在麻省理工学院Barbara Imperiali教授小组做博士后的头一年半时间里，我开发了一种免疫蛋白Im7的半合成途径，这在附件的研究总结中有详细的描述，这在以前被用作详细的蛋白质折叠研究的模型。这种半合成途径为获得免疫蛋白的新变体提供了无与伦比的机会，因此，最终可能导致对蛋白质折叠基本问题的新见解。沿着这些思路，我们合成了一种Im7的同质糖蛋白变体，首次研究了蛋白质糖基化对蛋白质折叠动力学的影响，并显示了合成的糖蛋白变体的不稳定性（比较研究摘要）。在这些结果的基础上，我们想提出免疫蛋白Im7的新变体，以进一步研究糖蛋白拓扑结构与特定蛋白质折叠行为之间的相关性。值得注意的是，我们打算使用精心设计的半合成路线，这需要在我博士后停留的第一阶段进行相当大的努力和大量的优化研究，以获得拟议的变体。

Recently peptide synthesis in combination with expressed protein ligation has been established as a powerful tool to access sufficient quantities of pure proteins for various biophysical and medicinal investigations. This combination of chemical and biochemical methods, in which a smaller synthetic peptide portion is connected to a larger expressed protein fragment via native chemical ligation, is particularly attractive, since it allows various modifications in the synthetic peptide portion leading to new variants of proteins which were previously inaccessible. Such modifications can include the incorporation of unnatural amino acids or conformationally constrained building blocks, the attachment of specific labels for monitoring activity or simply the alteration of whole domains in the protein sequence. Within the first one and a half years of my postdoctoral stay in the group of Prof. Barbara Imperiali at MIT, I was able to develop, as described in detail in the attached research summary, a semi-synthetic route to the immunity protein Im7, which has been used previously as a model for detailed protein folding studies. This semi-synthetic route represents an unparalleled opportunity to access new variants of immunity proteins and thus, ultimately could lead to new insights into fundamental questions concerning protein folding. Along these lines, we have synthesized a homogenous glycoprotein variant of Im7, which allowed the first study of the influence of protein glycosylation an the kinetics of protein folding and showed a destabilization for the synthesized glycoprotein variant (compare research summary). Building upon these results, we would like to propose new variants of the immunity protein Im7 to further investigate the correlation between the glycoprotein topology and the specific protein folding behaviour. lt is important to note that we intend to use the elaborated semi-synthetic route, which required considerable effort and numerous optimization studies within the first phase of my postdoctoral stay, for the access of the proposed variants.