Biophysical Characterization Of Macromolecules

在上一次报告所述期间，我们与大卫·马古利斯博士就构成多肽负载复合体的蛋白质之间的相互作用进行了合作。这是继续和扩大的。具体地说，我们研究了Tapasin以及TAPBPR(TAP结合蛋白相关)蛋白与MHC I类分子在负载不同多肽时相互作用的化学计量比和亲和力。虽然在目前的状态下主要使用分析性超速离心法，但该项目可能会演变为多蛋白质相互作用的全球多方法分析的模型应用。 在与Mark Mayer博士合作的另一个项目中，我们继续研究不同亚型的谷氨酸受体氨基末端结构域的同种和异种寡聚。这些受体的四级结构控制着它们的离子门控特性。我们进一步发展了研究高亲和力相互作用与荧光检测的沉降速度的必要方法。为了进一步扩大我们在荧光检测沉降速度的实际应用方面的经验，我们还与Tanja Mittag博士就SPOP蛋白质的自结合进行了合作研究。 我们已经开始与Patrizia Farci博士合作，应用表面等离子共振生物传感技术来检测从乙肝相关性急性肝功能衰竭患者的肝脏中分离和克隆的抗核心抗体与他们的乙肝核心抗原的相互作用。对这些抗体针对同源核心和野生型的结合表位、亲和力和动力学的测定，可能会对这些抗体在ALF发病机制中的作用提供新的见解。 最后，继续我们与Lawrence Samelson博士在T细胞激活后信号颗粒中多蛋白质相互作用的研究方面的长期合作，我们开展了旨在组装四组分适配蛋白质复合体的初步实验，并利用多信号沉降速度和量热技术研究它们的化学计量和亲和力。

In the last reporting period we have pursued a collaboration with Dr. David Margulies on the characterization of interactions of proteins constituting the peptide loading complex. This was continued and expanded. Specifically, we examined the stoichiometry and affinity of the interactions between tapasin, as well as TAPBPR (TAP binding protein-related) protein, with MHC class I molecules when loaded with different peptides. While largely employing analytical ultracentrifugation at the current state, this project may potentially evolve into a model application for global multi-method analysis of multi-protein interactions. In a separate project in collaboration with Dr. Mark Mayer, we have continued to study the homo- and hetero-oligomerization of different isoforms of glutamate receptor amino terminal domains. The quaternary structure of these receptors controls their ion gating properties. We have further developed the necessary methodology for studying high-affinity interactions with fluorescence-detected sedimentation velocity. In order to further broaden our experience with the practical application of fluorescence-detected sedimentation velocity, we have also carried out a collaborative study with Dr. Tanja Mittag on the self-association of SPOP protein. We have embarked on a collaboration with Dr. Patrizia Farci applying surface plasmon resonance biosensing to examine the interaction of anti-core antibodies isolated and cloned from livers of patients HBV-associated acute liver failure with their HBV core antigens. The determination of binding epitopes, affinities and kinetics of these antibodies against the homologous core and wild type may provide new insights into the role of these antibodies in the pathogenesis of ALF. Finally, continuing our long-term collaboration with Dr. Lawrence Samelson on the study of multi-protein interactions in signaling particles after T-cell activation, we have carried out initial experiments aimed at the assembly of four-component complexes of adapter proteins, and the study of their stoichiometries and affinities by multi-signal sedimentation velocity and calorimetric techniques.