Volume profile of biochemically responsive interfaces

一些蛋白质在配基结合时会发生主要的构象变化，如钙调素。在这个拟议的项目中，这一功能应转移到界面结构。水-固界面可通过多层或刷状的含有钙调蛋白的聚电解质或通过钙调蛋白突变体与固相的共价键合来修饰。通过这种方式，形成了与众所周知的对pH或温度敏感的界面结构不同的新型生物化学响应性界面结构。这种界面与许多应用相关，例如，需要对酶的固定化、细胞生长或药物释放进行调控。拟议项目的主要目标是控制这种界面层的建立，并表征这些层中的钙调蛋白功能。压力被用作关键变量来确定与界面层的建立、蛋白质的吸附以及与钙调蛋白结合的配体相关的体积变化。此外，生物化学响应界面的功能性作为压力的函数被研究，因为压力允许调节蛋白质和聚电解质之间的分子间相互作用和调节界面层的堆积密度。第一个资助期的成果和使用的方法，在那里已经调查了类似系统中的酶反应的压力效应，将为新提议的实验提供有益的基础。将应用高压表面敏感分析工具，并制备类似的界面修饰，辅之以钙调蛋白突变体的共价结合。此外，在第一轮实验中已经确定了对酶反应具有生物相容性的表面修饰。这一新项目的结果将详细描述钙调蛋白修饰的界面结构的组成、热力学和动力学性质以及生化功能，这对于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.