Quantifying Charge and Stability Influences on Protein Adsorption with Synthetic Mutants of Bacteriophage T4 Lysozyme

这个项目涉及一个动力学模型的应用，该模型描述了蛋白质在界面经历的一般事件序列，以量化蛋白质电荷和稳定性对界面行为的影响。噬菌体T4溶菌酶的选定合成突变体是从俄勒冈大学分子生物学研究所获得的转化细胞产生的。表现出热力学稳定性和电荷差异的突变体被用来量化这些分子性质对描述蛋白质在一系列疏水性不同的固体表面上的附着和展开的反应速率常数的影响。用二氯二甲基硅烷对硅表面进行硅烷化修饰，并用原位椭偏仪测量吸附动力学和描述每个特定蛋白质-表面接触的等温线。在该项目完成后，可以通过研究溶菌酶突变体混合物的吸附来重新检查和完善现有的多蛋白质吸附模型。然后，用更相关的蛋白质混合物进行实验，将允许与预测行为的偏差以更容易量化的方式与分子大小和形状等性质相关。这种方法有望为定量复杂介质中的竞争吸附提供一个良好的起点，并可能导致新的分离技术。

This project deals with application of a kinetic model describing the general sequence of events undergone by protein at an interface to quantify protein charge and stability effects on interfacial behavior. Selected synthetic mutants of bacteriophage T4 lysozyme are produced form transformed cells obtained from the Institute of Molecular Biology at the University of Oregon. Mutants exhibiting differences in thermodynamic stability and charge are used to quantify the influences of these molecular properties on reaction rate constants describing protein attachment and unfolding at each of a series of solid surfaces differing in hydrophobicity. Silicon surfaces will be modified by silanization with dichlorodimethylsilane, and in situ ellipsometry will be used to measure the adsorption kinetics and isotherm describing each specific protein- surface contact. Upon completion of the project, current multi-protein adsorption models could be re-examined and refined by studying adsorption from mixtures of lysozyme mutants. Experimentation with more relevant mixtures of proteins would then allow deviations from predicted behavior to be related to properties such as molecular size and shape in a more readily quantifiable way. This approach is expected to result in a sound starting point for quantifying competitive adsorption from complex media and may lead to new separation techniques.