SOLID STATE STRUCTURAL ANALYSIS OF SALIVARY PROTEINS

唾液蛋白的固态结构分析

• 批准号: 3425529
• 负责人: RONALD E LOOMIS
• 金额: $ 2.29万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3425529
• 关键词: biological models; chemical models; chemical structure function; computer simulation; hydroxyapatites; macromolecule; mathematical model; molecular dynamics; molecular shape; nuclear magnetic resonance spectroscopy; pellicle; protein structure function; saliva; solid state; solutions

The broad and long-term objective of the proposed study is to elucidate the physical-chemical and conformational basis for the biological functions of salivary molecules bound to tooth-like model surfaces. These types of investigations are required if one is to understand the molecular mechanisms responsible for the biological activities of salivary molecules in determining health nad disease. The specific aim of the current proposal is to obtain data from cross-polarization magic angle spinning nuclear magnetic resonance spectroscopy (CP/MAS NMR) on a well- characterized salivary peptide model system. Specifically, the multiply occurring nonapeptide segment (NH2-G-P-P-P-H-P-G-K-P-COOH or "PRG9"), derived from the proline-rich glycoprotein (PRG) found in human parotid saliva, will be used for this purpose. PRG9 is known to occur in the acquired enamel pellicle, binds calcium and acts as one of the primary structural determinants in native PRG. Also, the major solution-state conformations and substrate-bound physical-chemical properties of PRG9 have already been discerned. In order to achieve the stated specific aim, the carbon-13 (C13) solution-state NMR spectra of solubilized PRG9 will first be obtained. Subsequently, the C13 CP/MAS NMR spectra of "free" of "unbound" solid PRG9 and hydroxyapatite-bound peptide will be collected. A novel and necessary peak assignment strategy to be used involves collecting spectra of all PRG9's pertinent "subfragments" sequentially, starting with the N-terminal dipeptide through the intact nonapeptide, such that a complete library of resonance locations will be constructed. This is required for two reasons. First, the solid-state CP/MAS NMR experiment removes dipolar couplings and prohibits the use of experiments commonly used in solution-state NMR for spectral peak assignment (e.g. off-resonance decoupling, heteronuclear mapping, etc.). Second, the absence of NH peaks from proline disallows sequence-specific resonance assignments of these residues in the solution-state. The subfragment peptides will have the following amino acid sequences: (i) NH2-G-P-COOH, (ii) NH2-G-P-P-COOH, (iii) NH2-G-P-P-P-COOH, (iv) NH2-G-P-P-P-H-COOH, (v) NH2-G-P-P-P-H-P- COOH,(vi) NH2-G-P-P-P-H-P-G-COOH, and (vii) NH2-G-P-P-P-H-P-G-K-COOH. The solution-state, "free" solid-state and hydroxyapatite-adsorbed peptide NMR spectra will then be compared to each other, as well as data found in the literature. Assessment of PRG9 conformation will be based upon published C12 NMR spectra of peptides in known informational states. These results will represent the first structural analyses of salivary peptides at atomic resolution in the solid-state, and provide information sa to the conformational changes occurring upon substrate binding.

拟议的研究的广泛和长期目标是阐明 物理化学和构象基础的生物学功能 唾液分子与牙齿样模型表面结合。 这些类型的 如果要了解分子，则需要进行调查 负责唾液分子生物活性的机制 在确定健康NAD疾病时。 电流的具体目的 建议是从交叉极化魔术角旋转获得数据 核磁共振光谱（CP/MAS NMR） 表征唾液肽模型系统。 具体而言，乘以 发生非肽段（NH2-G-P-P-P-P-P-H-P-G-K-P-COOH或“ PRG9”）， 源自在人腮腺中发现的富含脯氨酸的糖蛋白（PRG） 唾液将用于此目的。 已知PRG9发生在 获得的搪瓷颗粒，结合钙并充当主要 本机PRG中的结构决定因素。 另外，主要解决方案 PRG9的构象和底物结合的物理化学特性 已经被辨别了。 为了达到既定目标， 碳-13（C13）溶液状态溶液溶液pRG9的NMR光谱将首先 获得。 随后，C13 CP/MAS NMR光谱的“免费” 将收集“未结合”固体PRG9和羟基磷灰石结合的肽。 一个 新颖和必要的高峰分配策略涉及收集 依次从 通过完整的非肽的N末端二肽，使得 将构建完整的共振位置库。 这是 需要两个原因。 首先，固态CP/MAS NMR实验 去除偶性耦合并禁止使用实验 用于溶液状态NMR用于光谱峰分配（例如非谐波 解耦，异核映射等）。 第二，不存在NH峰 从脯氨酸中删除这些序列特异性共振分配 溶液状态中的残留物。 亚纹理肽将具有 以下氨基酸序列：（i）NH2-G-P-COOH，（ii）NH2-G-P-P-COOH， （iii）NH2-G-P-P-P-P-COOH，（IV）NH2-G-P-P-P-P-P-H-COOH，（V）NH2-G-P-P-P-P-P-P-P-P-P- COOH，（VI）NH2-G-P-P-P-P-H-P-G-COOH和（VII）NH2-G-P-P-P-P-P-P-H-P-G-G-K-COOH。 这 溶液状态，“游离”固态和羟基磷灰石吸附的肽NMR 然后将比较光谱，以及在 文学。 PRG9构象的评估将基于发布 已知信息状态中肽的C12 NMR光谱。 这些结果 将代表原子唾液肽的第一个结构分析 在固态中解决，并向 底物结合时发生的构象变化。