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.

拟议研究的广泛和长期目标是澄清 生物功能的物理化学和构象基础 唾液分子结合在牙齿状的模型表面。这些类型的 如果一个人想要理解分子，就需要进行研究 唾液分子生物活性的作用机制 在确定健康和非传染性疾病方面。当前的具体目标是 建议从交叉极化魔角旋转中获取数据 一口井的核磁共振波谱(CP/MAS核磁共振) 特色化唾液肽模型系统。具体地说，乘法 出现非肽片段(NH2-G-P-H-P-G-K-P-COOH或“PRG9”)， 来源于人类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 获得性釉质膜，结合钙并作为原始层之一 原生PRG的结构决定因素。此外，主要的解决方案-国家 PRG9的构象和底物结合的物理化学性质 已经被识破了。为了实现所述的具体目标， 溶解的PRG9的碳-13(C13)固溶态核磁共振谱将首先 将被获得。随后，用~(13)CP/MAS核磁共振谱测定了其游离态。 未结合的固体PRG9和羟基磷灰石结合肽将被收集。一个 要使用的新颖且必要的峰值分配策略涉及收集 所有PRG9‘S相关“亚片段”的光谱顺序，从 通过完整的九肽的N-末端二肽，使得一个 将构建完整的共振位置文库。这是 这是必需的，原因有二。第一，固态CP/MAS核磁共振实验 删除偶极耦合并通常禁止使用实验 在溶液状态核磁共振中用于谱峰分配(例如，非共振 去偶联、异核映射等)。第二，没有NH峰 从脯氨酸不允许序列特定的共振分配这些 溶液状态下的残留物。亚片段多肽将具有 以下氨基酸序列：(I)NH2-G-P-COOH，(Ii)NH2-G-P-P-COOH， (Iii)NH2-G-P-COOH，(Iv)NH2-G-P-H-COOH，(V)NH2-G-P-H-P- (Vi)NH2-G-P-H-P-G-COOH和(Vii)NH2-G-P-H-P-G-K-COOH。这个 溶液状态、“游离”固态和羟基磷灰石吸附的多肽核磁共振 然后光谱将被相互比较，以及在 文学。对PRG9符合性的评估将基于发布的 已知信息态多肽的C12核磁共振波谱。这些结果 将首次在原子水平上对唾液肽进行结构分析 固态分辨率，并将信息sa提供给 底物结合时发生的构象变化。