D-Protein and D-Peptide Technology in Applied Research

化学蛋白质合成提供了获得整个D-蛋白质对映体的途径，在应用研究中具有独特的实用性。首先，蛋白质可以从L-和D-对映异构体的外消旋混合物中结晶，称为外消旋蛋白质晶体学，提供了容易获得高分辨率X射线结构的途径。为了了解膜活性细菌素是如何获得其抗菌特性的，本文对两种主要代表性细菌素--金色菌素a53（AucA）和乳酸菌素Q（LnqQ）进行了基于外消旋蛋白晶体学（0.9-1.2 μ m）的分析。通过结构分析和系统的残基取代，保守的表面修饰蛋白似乎在脂磷酸的配位中起重要作用，并且对抗菌活性至关重要。AucA中的额外色氨酸也参与形成低聚组装以增强稳定性。总的来说，外消旋蛋白质晶体学揭示了细菌素的分子相互作用，展示了细菌素如何提供其抗菌特性。其次，蛋白质药物靶标的D-对映体可用于镜像噬菌体展示（MIPD），允许发现非蛋白水解D-肽配体作为先导候选物。开发能够靶向肿瘤坏死因子受体I（TNFR 1）激活的D肽可能是控制由细胞因子风暴引起的炎症的有益解决方案。然而，含有144个残基和12个二硫键，MIPD的D-对映体的合成将是一个重大的挑战。在这里，分离的中链，细胞因子结合结构域（TNRCD 2）的报告，其对映异构体使MIPD发现一个低微摩尔亲和力的环肽结合剂。此外，还探索了使用计算方法来开发仅基于靶结构的D-蛋白配体。

Chemical protein synthesis provides access to entire D-protein enantiomers, with unique utility in applied research. Firstly, proteins may be crystallized from a racemic mixture of L- and D-enantiomers, known as racemic protein crystallography, providing facile access to high resolution X-ray structures. In this thesis, to understand how membrane-active bacteriocins have acquired their antibacterial properties, an analysis based on racemic protein crystallography (0.9-1.2 Å) of two key representatives, aureocin a53 (AucA) and lacticin Q (LnqQ) was conducted. Through structural analysis and systematic residue substitutions, conserved surface tryptophans appeared to play important roles in coordination of lipid phosphate and were critical for antibacterial activity. Additional tryptophan's in AucA were also involved in forming an oligomeric assembly for stability enhancement. Collectively, racemic protein crystallography shed light on the molecular interactions of tryptophans, demonstrating how a bacteriocin delivers its antibacterial properties. Secondly, D-enantiomers of protein drug targets can be used in mirror-image phage display (MIPD), allowing discovery of non-proteolytic D-peptide ligands as lead candidates. Development of a D-peptide capable of targeting tumor necrosis factor receptor I (TNFR1) activation could be a beneficial solution in controlling inflammation caused by cytokine storm. However, containing 144 residues and 12 disulfide bonds, synthesis of the D-enantiomer for MIPD would be a significant challenge. Here, isolation of the mid-chain, cytokine binding domain (TNRCD2) is reported, and its enantiomer enabled MIPD to discover a cyclic peptide binder with low micromolar affinity. Additionally, the use of computational methods to develop D-protein ligands based only on the target structure was explored.