DESIGN AND FUNCTION OF SALIVARY CYCLONEOGLYCOPEPTIDE

这项建议的长期目标是设计和加强 施他林的润滑作用与其相应的减量 细菌粘附性。具体地说，富含15N的史泰林将 以pGEX-2T为载体在优化的大肠杆菌表达系统中表达 克隆车。纯化的15N富集物的溶液结构 他汀类药物将使用三维核磁进行测定 共振光谱学。荧光和傅里叶变换红外光谱将用于 测试他汀类蛋白在结合时是否发生构象变化 羟基磷灰石。从这些生物物理研究中获得的信息将 与分子模拟一起用来推断三维结构 在牙釉质界面上的粘结剂。在界面上使用模型， 将选择特定的残基和二级结构基序用于 使用定点突变进行替换。假设是这样的 Statherin需要一个高极性的N-末端区域来锚定自己 接口和C端段，用于润滑性以及 细菌附着。他汀类药物的润滑性能取决于 基于其结合到牙釉质上时提供两亲性膜的能力 浮出水面。这一假设将通过专门准备几个 斯塔瑟林的类似物，其中C-末端区域的疏水性 通过将酪氨酸残基分别替换为更多 疏水残基，如Phe、Val、Ile和Leu。所有这些类比都将 是通过定点突变产生的。类似物的功效 增强润滑性，降低附着性 粘性放线菌到类似物包覆的羟基磷灰石将被检测。 来自这些类比行为的信息将允许设计和 具有最佳润滑性但细菌最少的模拟物的生产 粘附性。

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.