SOLID STATE STRUCTURAL ANALYSIS OF SALIVARY PROTEINS

拟议研究的广泛和长期目标是阐明 生物学功能的物理化学和构象基础 唾液分子附着在牙齿模型表面。 这些类型的 如果要了解分子， 负责唾液分子生物活性的机制 健康与疾病的关系 当前的具体目标 建议从交叉极化魔角旋转获得数据 核磁共振谱（CP/MAS NMR）， 特征的唾液肽模型系统。 具体来说，乘法 存在的九肽片段（NH 2-G-P-P-P-H-P-G-K-P-COOH或“PRG 9”）， 来源于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PRG 9发生在 获得性釉质表膜，结合钙，并作为一个主要的 天然PRG中的结构决定因素。 此外，主要的解决方案状态 PRG 9的构象和底物结合的物理化学性质具有 已经被发现了。 为了实现上述具体目标， 溶解的PRG 9的碳-13（C13）溶液状态NMR谱将首先 获得。 随后，使用C13 CP/MAS NMR光谱分析“不含”的化合物。 将收集“未结合的”固体PRG 9和羟基磷灰石结合的肽。 一 要使用的新颖且必要的峰分配策略涉及收集 所有PRG 9的相关“亚片段”的光谱顺序，从 N-末端二肽通过完整的九肽，使得 将构建谐振位置的完整库。 这是 有两个原因。 首先，固态CP/MAS NMR实验 消除偶极耦合，并禁止使用实验通常 在溶液状态NMR中用于光谱峰归属（例如，非共振 去耦、异源映射等）。 第二，没有NH峰 从脯氨酸不允许这些序列特异性共振分配 溶液状态下的残留物。 亚片段肽将具有 以下氨基酸序列：（i）NH 2-G-P-COOH，（ii）NH 2-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- （vi）NH2-G-P-P-P-H-P-G-COOH，和（vii）NH2-G-P-P-P-H-P-G-K-COOH。 的 溶液状态、“游离”固态和羟基磷灰石吸附肽NMR 然后将光谱相互比较，以及在 文学 PRG 9构象的评估将基于已发表的 已知信息状态下肽的C12 NMR谱。 这些结果 将代表唾液肽在原子水平上的首次结构分析， 在固态分辨率，并提供信息SA的 底物结合时发生的构象变化。

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.