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

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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.