Net charge in protein structure and aggregation: a charge ladder approach

描述（由申请人提供）：本提案描述了正在进行的和未来计划的研究，旨在确定净电荷在生物物理学相关问题中所起的作用。第一个目标提出了一个假设，即蛋白质的净电荷在决定蛋白质中酰胺氢交换速率方面起着重要作用-这是一个由pH为bbbb4的氢氧阴离子催化的反应。蛋白质电荷阶梯和质谱被用来测试这种电荷依赖的交换假设。这个提议的第二个目的是假设，与许多人类疾病相关的蛋白质聚集可以通过细微的化学修饰来抑制，这种化学修饰可以增加蛋白质的净电荷。控制电荷——而不是其他一些参数或性质，如天然状态的稳定性或结构——在开发药物治疗与蛋白质聚集有关的一系列疾病的过程中，是一个未被重视和未被探索的可能性。目的2中提出的实验旨在证明这种方法的概念（称为PRO-CHAIN；蛋白质聚集抑制）。目的3提出了实验来探索蛋白质和表面之间的静电相互作用是否可以用于i)促进ii)直接iii)模式，或iv)抑制蛋白质聚集到电荷微模式自组装单层（SAMs）上。蛋白质聚集在带电荷的表面，如脂质膜上，作为疾病发病机制的一种可能的情况正在被人们所接受。第三个目标描述了使用蛋白质电荷阶梯、微接触印刷和自组装单层的实验，以探索这一被怀疑与多种疾病相关的重要现象。与公共卫生的相关性：本提案与公共卫生直接相关；首先，它描述了一种开发药物治疗的全新方法，称为PRO-CHAIN（蛋白质聚集抑制电荷），它可能有助于开发治疗多种蛋白质聚集疾病的疗法，如阿尔茨海默病和II型糖尿病。此外，该提案描述的研究可能会进一步加深我们对蛋白质折叠研究中氢交换的分子决定因素的理解，其中许多研究都集中在导致人类疾病的致病性蛋白质上。最后，该建议使用新的尖端技术来研究可能与许多神经退行性疾病以及癌症相关的蛋白质表面相互作用。

DESCRIPTION (provided by applicant): This proposal describes ongoing and future-planned investigations that seek to determine the role that net charge plays in biophysics-related problems. The first aim presents the hypothesis that a protein's net charge plays a significant role in determining the rate of amide hydrogen exchange in proteins - a reaction that is catalyzed by hydroxide anion at pH>4. Protein charge ladders and mass spectrometry are used to test this charge-dependent exchange hypothesis. The second aim of this proposal hypothesizes that protein aggregation, as related to many human diseases, can be inhibited with subtle chemical modifications that increase the protein's net charge. Controlling charge - as opposed to some other parameter or property such as native-state stability or structure - is an underemphasized and unexplored possibility in developing drug therapies for a wide array of diseases linked to protein aggregation. The experiments proposed in Aim 2 are intended as a proof of concept for this approach (termed PRO-CHAIN; PROtein CHarging for Aggregation INhibition). Aim 3 proposes experiments to explore if electrostatic interactions between proteins and surfaces can be used to i) promote ii) direct iii) pattern, or iv) inhibit protein aggregation onto charge- micro-patterned self assembled monolayers (SAMs). The aggregation of proteins onto charged surfaces, such as lipid membranes, is gaining acceptance as a likely scenario in disease pathogenesis. This third aim describes experiments using protein charge ladders, micro-contact printing and self assembled monolayers to explore this important phenomenon that is suspected to be relevant to a wide range of diseases. Relevance to public health: This proposal has immediate relevance to public health; first, it describes a whole new approach for developing drug therapies, referred to as PRO-CHAIN (PROtein CHarging for Aggregation INhibition), that may aid in the development therapies for the treatment of a wide range of protein aggregation diseases such as Alzheimer's disease and type II diabetes. Furthermore, this proposal describes research that may further our understanding of the molecular determinants of hydrogen exchange in protein folding studies -- many of these studies focus on pathogenic proteins that cause human disease. Lastly, this proposal uses new cutting edge technologies to study protein-surface interactions of the sort that may be related to numerous neurodegenerative diseases, as well as cancer.