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

最近，肽合成与表达的蛋白质连接的组合已被确立为获得足够量的纯蛋白质用于各种生物物理和医学研究的有力工具。这种化学和生物化学方法的组合，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，是特别有吸引力的，因为它允许合成肽部分的各种修饰，导致以前无法获得的蛋白质的新变体。这样的修饰可以包括掺入非天然氨基酸或构象受限的结构单元、连接用于监测活性的特异性标记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的Barbara Imperiali教授小组做博士后的前一年半里，我能够开发出免疫蛋白Im7的半合成路线，如所附研究摘要中详细描述的那样，Im7以前曾被用作详细蛋白质折叠研究的模型。这种半合成路线代表了获得免疫蛋白新变体的无与伦比的机会，因此最终可能导致对蛋白质折叠基本问题的新见解。沿着这些路线，我们合成了Im7的同质糖蛋白变体，这允许首次研究蛋白糖基化的影响和蛋白折叠的动力学，并显示合成的糖蛋白变体的不稳定性（比较研究总结）。基于这些结果，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和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，这需要相当大的努力和大量的优化研究，在我的博士后停留的第一阶段，为所提出的变体的访问。

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.