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

描述(由申请人提供)：这项提案描述了正在进行的和未来计划的调查，旨在确定净电荷在生物物理学相关问题中所起的作用。第一个目标提出的假设是，蛋白质的净电荷在决定蛋白质中的酰胺氢交换速率方面起着重要作用--这是一种在pH为4的条件下由氢氧阴离子催化的反应。蛋白质电荷梯和质谱学被用来检验这一依赖于电荷的交换假说。这项提议的第二个目的假设，与许多人类疾病有关的蛋白质聚集可以通过微妙的化学修饰来抑制，从而增加蛋白质的净电荷。在开发与蛋白质聚集相关的广泛疾病的药物疗法时，控制电荷--而不是一些其他参数或性质，如天然状态稳定性或结构--是一种未被重视和探索的可能性。目标2中提出的实验旨在证明这一方法的概念(称为PRO-Chain；蛋白质充电抑制聚集)。目的3提出实验以探索蛋白质和表面之间的静电相互作用是否可以被用来促进II)直接III)模式，或IV)抑制蛋白质在电荷微图案化自组装单层(SAM)上的聚集。蛋白质聚集在带电表面，如脂膜，正在被认为是疾病发病机制中的一种可能的情景。第三个目标描述了使用蛋白质电荷梯子、微接触打印和自组装单层膜来探索这一被怀疑与一系列疾病有关的重要现象的实验。与公共健康的相关性：这项建议与公共健康直接相关；首先，它描述了一种全新的药物疗法开发方法，称为PRO-Chain(抑制聚集的蛋白质充电)，可能有助于开发治疗多种蛋白质聚集性疾病的疗法，如阿尔茨海默病和II型糖尿病。此外，这一建议描述了可能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的研究--许多这些研究侧重于导致人类疾病的致病蛋白质。最后，这项建议使用新的尖端技术来研究蛋白质-表面相互作用，这种相互作用可能与许多神经退行性疾病和癌症有关。

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.