Volume profile of biochemically responsive interfaces

一些蛋白质在配体结合时发生重大构象变化，如钙调蛋白。此功能应在此拟议项目中转移到界面结构中。水-固界面可以通过含有钙调蛋白的多层或刷状聚电解质或钙调蛋白突变体与固相的共价键来修饰。通过这种方式，形成了新的生化反应界面结构，与众所周知的pH或温度敏感的界面结构形成对比。这种界面与许多应用相关，例如，酶的固定化，细胞生长或药物的释放需要调节。该项目的主要目标是控制这些界面层的形成，并表征这些层中的钙调素功能。压力是决定与界面层的形成、蛋白质的吸附和配体与钙调素结合相关的体积变化的关键变量。此外，生物化学反应界面的功能作为压力的函数进行了研究，因为压力可以调节蛋白质和聚电解质之间的分子间相互作用，并调节界面层的堆积密度。在第一个资助阶段，研究了类似系统中压力对酶反应的影响，所取得的成果和使用的方法将为新提出的实验提供有益的基础。高压表面敏感分析工具将被应用，类似的界面修饰也将被制备，辅之以钙调素突变体的共价结合。此外，在第一轮实验中已经确定了具有酶反应生物相容性的表面修饰。这个新项目的结果将详细描述钙调素修饰的界面结构的构建、热力学和动力学性质以及生化功能，这对理解这些系统及其生物技术和生物医学应用非常重要。

Some proteins undergo major conformational changes upon ligand binding, such as calmodulin. This functionality shall be transferred to interfacial structures in this proposed project. Aqueous-solid interfaces are modified by multilayers or brushes of polyelectrolytes incorporating calmodulin or by covalent bonding of calmodulin mutants to the solid phase. In this way, novel biochemically responsive interfacial structures are formed, in contrast to well-known pH or temperature sensitive interfacial architectures. Such interfaces are relevant for many applications, where, for example, the immobilization of enzymes, the cell growth or the release of drugs needs to be regulated. Main goals of the proposed project are the controlled build-up of such interfacial layers and the characterization of the calmodulin functionality in these layers. Pressure is used as key variable to determine the volume changes associated with the build-up of the interfacial layers, the adsorption of protein and the ligand binding to calmodulin. Furthermore, the functionality of the biochemically responsive interfaces is investigated as a function of pressure, because pressure allows for tuning intermolecular interactions between protein and polyelectrolyte and modulating the packing density of the interfacial layers. The achievements and the methods used in the first funding period, where pressure effects on enzyme reactions in similar systems have been investigated, will be a helpful basis for the new proposed experiments. High-pressure surface-sensitive analytical tools will be applied, and similar interfacial modifications are prepared, complemented by covalent binding of calmodulin mutants. Moreover, surface modifications, which are biocompatible for enzyme reactions, have been identified in the first round of experiments. The results of this new project will describe the build-up, the thermodynamic and dynamic properties as well as the biochemical functionality of calmodulin-modified interfacial structures in detail, which is important for the understanding of these systems and their biotechnological and biomedical applications.