STRUCTURE-FUNCTION ANALYSIS OF INTESTINAL FATTY ACID-BIN

肠脂肪酸仓的结构功能分析

• 批准号: 6188487
• 负责人: Judith Storch
• 金额: $ 4.03万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6188487
• 关键词: fatty acid binding protein; fluorescence resonance energy transfer; hydropathy; intestines; lipid bilayer membrane; membrane activity; membrane permeability; protein localization; protein structure function; site directed mutagenesis; tissue mosaicism

Intestinal enterocytes express high levels of two homologous fatty acid-binding proteins (FABP), liver FABP (LFABP) and intestinal FABP (IFABP). It is hypothesized that FABPs are important in intracellular transport of FA. Both I- and LFABP bind long chain fatty acids (FA) with high affinity, and it has recently been shown that they have similar tertiary structures. Nevertheless, it has been suggested that they are functionally distinct because they have different substrate specificities and stoichometry and different tissue distributions. Furthermore, recent work from our laboratory has shown that these proteins employ markedly different mechanisms to transfer FA to acceptor membranes. Whereas FA transfer from LFABP occurs via aqueous diffusion, FA transfer from IFABP occurs during direct collisional interactions of the protein with acceptor membranes. We have recently demonstrated the critical importance of the helix-turn helix domain in the interaction of IFABP with membranes, and in its FA transfer mechanism. In the present proposal we will use biochemical, molecular biological and biophysical techniques to provide a molecular level analysis of the specific domains and residues that are involved in these IFABP-membrane interactions, as well as a detailed understanding of the physicochemical nature of the IFABP-membrane association itself. Further, the structural basis for the absence of collisional transfer of FA from LFABP will also be elucidated. The specific aims of this proposal are: 1) To determine the importance of the alphaI helix and the alphaII helix in collisional transfer of fatty acids to membranes, by constructing chimeric I/LFABP; and 2) To elucidate the roles of specific charged and hydrophobic residues in collisional transfer of FA to membranes, employing site-directed mutagenesis techniques. This approach to the fatty acid transport function of FABPs should provide molecular level detail of the specific structural elements which lead to the unique transport properties of IFABP versus LFABP. Specifically, for all point mutant and chimeric proteins engineered, we will analyze the rate and mechanism of fatty acid transfer to model membranes using a fluorescence resonance energy transfer assay, and the interaction of the proteins with membranes using several complementary biochemical and biophysical techniques. Since both LFABP and IFABP are highly expressed in the small intestinal enterocyte, an understanding of their mechanisms of actions will enable the effective nutritional and/or pharmacological manipulation of dietary lipid assimilation.

肠肠细胞表达高水平的两种同源脂肪酸结合蛋白（FABP），肝脏FABP（LFABP）和肠道FABP（IFABP）。 假设FABP在FA的细胞内运输中很重要。 I-和LFABP都结合了具有高亲和力的长链脂肪酸（FA），并且最近证明它们具有相似的三级结构。然而，已经提出它们在功能上是不同的，因为它们具有不同的底物特异性和化学计和不同的组织分布。 此外，我们实验室的最新工作表明，这些蛋白质采用明显不同的机制将FA转移到受体膜上。 尽管FA从LFABP转移是通过水扩散发生的，而从IFABP的FA转移在蛋白质与受体膜的直接碰撞相互作用期间发生。最近，我们证明了螺旋 - 螺旋螺旋结构域在IFABP与膜的相互作用及其FA转移机制中的重要性。 在本提案中，我们将使用生化，分子生物学和生物物理技术来提供对这些IFABP-膜相互作用所涉及的特定域和残基的分子水平分析，以及对IFABP-MEMBRANE联想本身的物理化学性质的详细理解。 此外，还将阐明不存在FA碰撞转移的结构基础。该提案的具体目的是：1）通过构建嵌合I/LFABP来确定脂肪酸向脂肪酸碰撞转移到膜上的碰撞转移中的重要性； 2）阐明采用位置定向的诱变技术，阐明了特异性带电和疏水残基在FA向膜转移中的作用。 Fabps脂肪酸传输功能的这种方法应提供特定结构元素的分子水平细节，从而导致IFABP与LFABP的独特传输特性。 具体而言，对于设计的所有点突变蛋白和嵌合蛋白，我们将使用荧光谐振能量转移测定法分析脂肪酸转移到对膜建模的速率和机制，以及使用几种互补的生物化学和生物物理技术的蛋白质与膜与膜的相互作用。 由于LFABP和IFABP均在小肠肠上皮细胞中高度表达，因此对它们的作用机制的理解将使饮食脂质同化的有效营养和/或药理学操纵。