PROTEIN DESIGN BY DYNAMIC COMBINATORIAL CHEMISTRY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本申请的长期目标是设计可因其高稳定性而离体选择的蛋白质。这样的蛋白质将对蛋白水解具有抗性，并且将耐受将降低其免疫原性的表面修饰。因此，它们将是需要破坏内源性蛋白质-蛋白质相互作用的治疗应用的理想候选物。成功完成拟议的研究将揭示赋予小球状蛋白质最佳折叠稳定性的氨基酸序列。体外化学组装和选择策略为随后的体内表达提供候选序列。具体地，使用交换不稳定的金属配体化学完成肽二级结构元件组装成预定折叠。在热力学控制下，亚基交换配偶体，分离出最稳定的缔合物，并确定其氨基酸序列。 基于使用合成三核苷酸从体外选择返回的序列信息构建DNA盒。过表达的蛋白质被分离和表征。特别令人感兴趣的是这些从头蛋白质的折叠稳定性，以及它们与类似结构的天然蛋白质相比的折叠动力学。创造具有极其稳定的折叠结构的蛋白质将为基于蛋白质的药物在小分子无效的治疗策略中的应用打开大门。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term objective of this application is to design proteins that can be selected ex-vivo for their high stability. Such proteins will be resistant to proteolysis, and will tolerate surface modifications that will reduce their immunogenicity. As such, they will be ideal candidates for therapeutic applications requiring the disruption of endogenous protein-protein interactions. Successful completion of the proposed research will reveal amino-acid sequences that confer optimal folding stability on small globular proteins. An in vitro chemical assembly and selection strategy provides candidate sequences for subsequent in vivo expression. Specifically, assembly of peptide secondary structural elements into a pre-determined fold is accomplished using exchange-labile metal-ligand chemistry. The subunits exchange partners under thermodynamic control, and the most stable associations are isolated and their amino acid sequences determined. DNA cassettes are constructed based on the sequence information returned from the in vitro selection using synthetic trinucleotides. Overexpressed proteins are isolated and characterized. Of particular interest are the folding stabilities of these de novo proteins, and their folding kinetics as compared with natural proteins of similar structure. The creation of proteins with extremely stable folded structures will open the door to protein-based drugs for applications in therapeutic strategies where small molecules are ineffective.