Protein folding, aggregation and engineering

蛋白质中氨基酸的顺序，即一级序列，编码蛋白质如何折叠成其功能性“天然”状态，以及它如何错误折叠形成非功能性和潜在毒性的聚集体。 尽管进行了广泛的研究，但一级序列决定蛋白质折叠、结构、动力学和功能的相互关联性质的分子机制远未被完全理解。 我们正在详细的原子水平上分析具有生物学、医学和生物技术重要性的蛋白质的这些性质。我们的实验方法是采用一系列生物化学和生物物理技术（如表达和工程重组蛋白的分子生物学方法，以及多维异构NMR光谱，光谱，化学变性，量热法，质谱，光散射和显微镜）来表征天然和工程蛋白质的性质。 本论文的主要内容包括三个方面：1）模式生物D. discoideum及其人类同系物fascin。 这些蛋白质在生物学和疾病中的细胞运动中具有重要作用。我们将研究它们的肌动蛋白结合和可逆膜结合的分子机制，这是基本的生物学重要性。 我们还将分析折叠和错误折叠形成包涵体聚集体的分子机制，这对于生物技术应用和理解蛋白质错误折叠疾病是有意义的; 2）由于超声处理导致蛋白质损伤和聚集的分子机制。这些研究对超声在研究、工业和医学中的许多常见应用具有重要意义; 3）YchN样蛋白的折叠和工程化，以阐明相关蛋白质中二级、三级和四级结构的决定因素，以及蛋白质结构进化的机制。 我们的研究将为这些蛋白质的重要特征提供基本见解，这些蛋白质将适用于许多其他蛋白质的理解，工程和设计。

The order of amino acids in a protein, i.e. the primary sequence, encodes how a protein folds into its functional "native" state, as well as how it may misfold to form nonfunctional and potentially toxic aggregates.  Despite extensive study, the molecular mechanisms by which primary sequence determines the inter-related properties of protein folding, structure, dynamics and function are far from being fully understood.  We are analyzing these properties at a detailed atomic level for proteins of biological, medical and biotechnological importance. Our experimental approach is to employ a battery of biochemical and biophysical techniques (such as molecular biological methods to express and engineer recombinant proteins, and multi-dimensional heteronuclear NMR spectroscopy, optical spectroscopies, chemical denaturation, calorimetry, mass spectrometry, light scattering and microscopy) to characterize the properties of natural and engineered proteins.       This proposal describes three projects: 1) folding and function of hisactophilin, an actin-bundling protein from the model organism, D. discoideum, and its human homolog, fascin.  These proteins have important roles in cell motility in biology and disease. We will study their molecular mechanisms of actin binding and reversible membrane binding, which are of fundamental biological importance.  We will also analyze molecular mechanisms of folding and of misfolding to form inclusion body aggregates, which are of interest for biotechnological applications and for understanding protein misfolding diseases; 2) molecular mechanisms of damage and aggregation of proteins due to sonication. These studies have important implications for many common applications of sonication in research, industry and medicine; and 3) folding and engineering of YchN-like proteins, to elucidate the determinants of secondary, tertiary and quaternary structure among related proteins, and mechanisms of protein structure evolution.  Our studies will provide fundamental insights into important characteristics of these proteins that will be applicable to the understanding, engineering and design of many other proteins.