The Folding Mechanism of the Intracellular Lipocalins

9405282 Ropson本研究的目的是检查细胞内脂质运载蛋白家族的几个成员的动力学折叠机制。 这些蛋白质共享显着的结构相似性的X射线晶体学，尽管只有20%的序列同一性。 这里提出的实验将确定具有相同结构的蛋白质是否也具有相同的折叠机制，即使它们的序列中存在低水平的同源性。 将采用多方面的办法在各级结构组织中解决这一问题。 停流圆二色性将用于检查折叠和展开过程中二级结构的整体变化。 此外，对于该家族中具有质子NMR分配的蛋白质，骨架酰胺-氢交换标记将用于确定何时形成二级结构并在折叠过程中稳定的特定氢键。 通过监测色氨酸荧光和用氟和氟NMR对蛋白质进行位点特异性标记，检查折叠过程中三级结构的形成。 这种独特的方法允许在特定的网站在折叠过程中三级接触的变化进行监测，无论是在平衡和动力学通过停流NMR。 氟核磁共振已经确定了一个不寻常的中间体，几乎没有任何二级结构的家庭成员，肠脂肪酸结合蛋白。 该中间体在足以通过其他光谱方法完全解折叠蛋白质的变性剂水平下在平衡时高度聚集。 这些研究将扩展到脂质运载蛋白家族的其他成员，以确定在其折叠途径上是否也存在相同类型的中间体。 蛋白质折叠的机制仍然是生物化学中一个难以捉摸的问题。 目前尚不清楚蛋白质的氨基酸序列如何决定蛋白质的最终结构以及在该结构形成期间可能存在的中间体的结构。 该建议使用多种物理方法来确定细胞内脂质运载蛋白家族蛋白质的折叠机制。 对这个蛋白质家族的研究将提高我们对蛋白质折叠的一般理解，特别是对折叠蛋白质的理解，这将有利于生物技术工业生产改进的蛋白质产品。 ***

9405282 Ropson The purpose of this study is to examine the kinetic folding mechanism of several members of the intracellular lipocalin family of proteins. These proteins share remarkable structural similarity by x-ray crystallography despite as little as 20% sequence identity. The experiments proposed here will determine if proteins that share the same structure also share the same folding mechanism, even when there are low levels of homology in their sequence. A multifaceted approach will be used to address this problem on all levels of structural organization. Stopped- flow circular dichroism will be used to examine global changes in secondary structure during folding and unfolding. In addition, for proteins in this family that have proton NMR assignments, backbone amide-hydrogen exchange labeling will be used to determine when the specific hydrogen bonds that form the secondary structure and stabilized during the folding process. Formation of tertiary structure during folding will be examined by monitoring tryptophan fluorescence and by site-specific labeling of the protein with fluorine and fluorine NMR. This unique method allows the changes in tertiary contacts at specific sites during folding to be monitored, both at equilibrium and kinetically by stopped-flow NMR. Fluorine NMR has already identified an unusual intermediate that has little if any secondary structure for one member of the family, intestinal fatty-acid binding protein. This intermediate is highly populated at equilibrium at levels of denaturant sufficient to completely unfold the protein by other spectroscopic methods. These studies will be extended to other members of the lipocalin family to determine if the same types of intermediates are present on their folding pathways as well. %%% The mechanism of protein folding continues to be a elusive problem in biochemistry. It is not currently known how the amino acid sequence of a protein determines the final structure of the protein and the structures of the intermediates that might be present during the formation of that structure. This proposal uses a variety of physical methods to determine the mechanism of folding for the intracellular lipocalin family of proteins. The study of this family of proteins will improve our understanding of protein folding in general, and of -sheet proteins in particular, which will have benefits for the production of improved protein products by the biotechnology industry. ***