Functionalized Gold Nanoparticles: Understanding the Mechanism of Protein Binding

描述(申请人提供)：功能化的金纳米颗粒(AuNPs)为靶向药物输送的挑战提供了一种有前途的解决方案，它们具有独特的光谱特性，有助于光学检测和诊断。已经设计了几种基于AuNP的系统来治疗癌症和神经退行性疾病，还设想了治疗传染病的方法。蛋白质分子自发地吸附在AuNP表面，设计功能化AuNPs的一种方法是利用这种自发过程直接结合活性酶。不幸的是，并不是所有的酶都在AuNP表面保持活性，目前还不可能预测一种酶在被吸附时是否会保持其活性。这是该领域的一个主要障碍，因此，通用的、蛋白质功能化的AuNPs仍然是一个遥远的目标。然而，在实现创造基于纳米颗粒的新疗法的长期目标之前，必须对AuNP表面的蛋白质结构有更好的理解。这种更好的理解是这项提议的直接目标。给出了初步的数据，证明了利用核磁共振波谱研究吸附蛋白质结构的可行性。核磁共振有可能在单个残基的分辨率上揭示结构细节，而不是最近的进展 在大分子核磁共振领域，还没有得到充分的利用。这项工作将创新的核磁共振方法应用于这一具有挑战性和重要的问题。具体地说，这项提议有三个具体目标：(1)确定AuNP-蛋白质相互作用背后的驱动残基。实现这一目标的方法是使用新开发的PULSE程序来研究蛋白质结合的初始阶段。(2)确定AuNP表面蛋白的结构和取向。在这一目标中，氢/氚交换方法将与侧链同位素标记相结合，以生成AuNP表面的结构图。(3)合成具有碳酸氢酶活性的功能化AuNPs。这一最终目标将开发一个通用的模型系统，可靠地功能化具有酶活性的AuNPs。通过这些目标，将更好地了解AuNPs上的蛋白质结构，并将开发出创建功能化AuNPs的一般设计原则。使用这里使用的核磁共振方法，将有可能检查吸附蛋白质的结构变形，并将更好地了解蛋白质在结合的初始阶段的行为。这些发现将产生积极影响，因为它们将使研究人员能够更快、更有效地开发基于AuNP的治疗和诊断方法，这将导致生物医学科学中基于纳米技术的工具明显更好。

DESCRIPTION (provided by applicant): Functionalized gold nanoparticles (AuNPs) offer a promising solution to the challenge of targeted drug delivery, and they possess unique spectroscopic properties that facilitate optical detection and diagnostics. Several AuNP- based systems have been designed to treat cancer and neurodegenerative diseases, and treatments are envisioned for infectious disease as well. Protein molecules spontaneously adsorb to AuNP surfaces, and one approach to designing functionalized AuNPs is to bind active enzymes directly using this spontaneous process. Unfortunately, not all enzymes remain active on the AuNP surface, and it is currently impossible to predict whether an enzyme will retain its activity when adsorbed. This represents a major hurdle for the field, and as a result general-purpose, protein-functionalized AuNPs remain a distant goal. Before the long-term goal of creating new nanoparticle-based therapies is realized, however, an improved understanding of protein structure on the AuNP surface must be developed. This improved understanding is the immediate objective of this proposal. Preliminary data is presented demonstrating the feasibility of using NMR spectroscopy to investigate the structure of adsorbed proteins. NMR has the potential to reveal structural detail at the resolution of individual residues, bet recent advances in large-molecule NMR have not been adequately leveraged in this field. This work applies innovative NMR methodologies to this challenging and important problem. Specifically, this proposal has three specific aims: (1) Identify the driving residues behind AuNP-protein interactions. The approach for this aim is to use newly-developed pulse programs to study the initial stages of protein association. (2) Determine the structure and orientation of AuNP surface proteins. In this aim, hydrogen/deuterium exchange methods will be combined with side-chain isotopic labeling to yield a picture of structure on the AuNP surface. (3) Synthesize functionalized AuNPs with carbonic anhydrase activity. This final aim will develop a general model system for reliably functionalizing AuNPs with enzymatic activity. Through these aims, a much better understanding of protein structure on AuNPs will be attained, and general design principles for creating functionalized AuNPs will be developed. Using the NMR approaches employed here, it will be possible to examine structural deformations of adsorbed proteins, and the behavior of proteins during the initial stages of binding will be better understood. These discoveries will have a positive impact because they will enable researchers to develop AuNP-based therapeutics and diagnostics much more quickly and efficiently, and this will lead to markedly better nanotechnology-based tools in the biomedical sciences.