DESIGN AND FUNCTION OF SALIVARY CYCLONEOGLYCOPEPTIDE

本提案的长期目标是设计和加强 他他林的润滑功能伴随着其 细菌粘附特性。 具体地，15 N富集的斯达林将 在优化的E.以pGEX-2 T为载体， 克隆车 纯化的15 N富集的溶液结构 将使用三维核磁共振测定Statherin 共振光谱学 荧光和FTIR光谱将用于 测试在结合后，他色林是否经历任何构象变化， 羟基磷灰石 从这些生物物理研究中获得的信息将 与分子建模一起使用来推断三维结构 在牙釉质界面上的他他宁。 使用界面处的模型， 将选择特定的残基和二级结构基序， 使用定点诱变进行取代。 前提是 雄甾苷肽需要高极性的N-末端区域以将其自身锚在 界面和C-末端段的润滑性，以及 细菌附着 司他林的润滑性能取决于 基于其在与牙釉质结合时提供两亲性膜的能力 面 这一假设将通过专门准备几个 其中C-末端区域的疏水性 将通过用更多的酪氨酸残基单独取代酪氨酸残基来增加 疏水残基如Phe、瓦尔、Ile和Leu。 所有这些类似物将 通过定点诱变产生。 类似物的功效， 增强润滑和减少附着的能力 将检查粘性放线菌与类似物涂层羟基磷灰石的关系。 来自这些类似物的行为的信息将允许设计和 生产具有最佳润滑但细菌最少的类似物 粘合性能

The long-term goal of this proposal is the design and enhancement of the lubricative function of statherin with a concomitant decrease of its bacterial adhesion properties. Specifically, 15N-enriched statherin will be expressed in an optimized E. coli expression system using pGEX-2T as the cloning vehicle. The solution structure of the purified 15N-enriched statherin will be determined using three-dimensional nuclear magnetic resonance spectroscopy. Fluorescence and FTIR spectroscopy will be used to test whether statherin undergoes any conformational change upon binding to hydroxyapatite. The information gained from these biophysical studies will be used with molecular modeling to deduce the three-dimensional structure of statherin at the enamel interface. Using the model at the interface, specific residues and secondary structural motifs will be selected for substitution using site-directed mutagenesis. The hypothesis is that statherin requires a highly polar N-terminal region to anchor itself at the interface and the C-terminal segment for both lubricity, as well as bacterial attachment. The lubrication property of statherin is dependent upon its ability to provide an amphipathic film upon binding to the enamel surface. This hypothesis will be tested by specifically preparing several analogs of statherin in which the hydrophobicity of the C-terminal region will be increased by individually replacing the tyrosine residues with more hydrophobic residues such as Phe, Val, Ile and Leu. All these analogs will be produced by site-directed mutagenesis. The efficacy of the analogs to enhance lubrication and their ability to decrease the attachment of Actinomyces viscosus to analog-coated hydroxyapatite will be examined. Information from the behavior of these analogs will allow the design and production of an analog with optimum lubrication but minimal bacterial adhesion properties.