Understanding and engineering hydrophobin self-assembly

蛋白质自组装在自然界中扮演着许多重要的角色，为生物材料的发展提供了新的机遇。一种自组装蛋白质家族是疏水素，它是丝状真菌为改变其环境而产生的小球状蛋白质。分泌的疏水素的行为就像洗涤剂，可以改变形状形成小棒，这是一种极其耐用的防水涂层，覆盖在气生菌丝和孢子上。疏水蛋白不同寻常的生化特性使人们有兴趣将其开发为商业用途，用作乳化剂、泡沫稳定剂以及保护和修饰表面的试剂。目前，对疏水素的性质以及它们如何形成RODLET缺乏全面的、基于分子的了解。目前只有少数几种疏水蛋白的高分辨结构，但这些结构针对的疏水蛋白在序列和结构上是如此的多样化，以至于很难将这些疏水蛋白的序列和结构特性与它们的功能联系起来。在与艾玛·马斯特博士(UofT)的合作下，我已经开始描述一种新的疏水蛋白亚类的结构和功能。通过对这些疏水蛋白的研究，我将把疏水蛋白的序列特征与它们的功能特性联系起来。具体地说，这项建议的目标是：1.表征不同疏水蛋白的结构和功能，并确定哪些因素影响它们的性质。2.确定小棒是如何形成的，并建立小棒结构的工作模型。3.设计具有所需特性的疏水素。为了实现这些目标，我将使用一种结合分子生物学、结构生物学和生物物理学的实验方法。这项工作将有助于我们对蛋白质自组装的知识，并将提供对疏水蛋白属性和功能的机械理解，这对将其用于商业应用至关重要。我的研究计划将允许学员发展分子生物学、核磁共振光谱学、X射线结晶学、圆二色光谱偏振法、原子力显微镜和电子显微镜的技能。这些不同的技能和经验将确保高素质的人员在生物技术、制药和学术部门未来的职业生涯中取得成功。

Protein self-assembly plays many essential roles in nature and presents new opportunities to develop biomaterials. One family of self-assembling proteins are hydrophobins, which are small globular proteins produced by filamentous fungi to modify their environment. Secreted hydrophobins behave like detergents and can change shape to form rodlets, which are an extremely durable and water repellent coating over aerial hyphae and spores. The unusual biochemical properties of hydrophobins have generated interest in developing them for commercial applications as emulsifiers, foam stabilizers and as an agent to protect and modify surfaces. Currently a comprehensive, molecular-based understanding of the properties of hydrophobins and how they form rodlets is lacking. Only a handful of high-resolution structures for hydrophobins exist, but these are for hydrophobins so diverse in sequence and structure that it is difficult to correlate the sequence and structural properties of these hydrophobins with their function. In collaboration with Dr. Emma Master (UofT) I have begun to characterize the structure and function of a new sub-class of hydrophobins. By studying these hydrophobins I will correlate sequence features of hydrophobins with their functional properties. Specifically, the objectives of this proposal are to: 1. Characterize the structure and function of diverse hydrophobins and determine what factors influence their properties. 2. Determine how rodlet formation occurs and build a working model of the rodlet structure. 3. Design hydrophobins that assemble into rodlets with desired properties.To address these objectives I will use an experimental approach combining molecular biology, structural biology, and biophysics. This work will contribute to our knowledge of protein self-assembly and will provide a mechanistic understanding of hydrophobin properties and function that is critical to engineer them for commercial applications. My research program will allow trainees to develop skills in molecular biology, nuclear magnetic resonance spectroscopy, X-ray crystallography, circular dichroism spectropolarimetry, atomic force microscopy, and electron microscopy. These diverse skills and and experience working with a protein that is currently being developed for commercialization will ensure the success of highly qualified personnel in their future careers in the biotechnology, pharmaceutical and academic sectors.