STRUCTURE-FUNCTION ANALYSIS OF INTESTINAL FATTY ACID-BIN

肠上皮细胞表达高水平的两种同源脂肪酸结合蛋白（FABP），肝FABP（LFABP）和肠FABP（IFABP）。 推测FABP在FA的细胞内转运中是重要的。 I-和LFABP都以高亲和力结合长链脂肪酸（FA），并且最近已经表明它们具有相似的三级结构。然而，有人认为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量学以及不同的组织分布。 此外，我们实验室最近的工作表明，这些蛋白质采用显着不同的机制来转移FA受体膜。 而FA从LFABP转移发生通过水扩散，FA从IFABP转移发生在蛋白质与受体膜的直接碰撞相互作用。我们最近已经证明了螺旋-转角螺旋结构域在IFABP与膜的相互作用及其FA转移机制中的至关重要性。 在本提案中，我们将使用生物化学，分子生物学和生物物理技术，以提供一个分子水平的分析的具体领域和残基，参与这些IFABP膜相互作用，以及详细了解IFABP膜协会本身的物理化学性质。 此外，从LFABP的FA碰撞转移的情况下的结构基础也将得到阐明。该建议的具体目的是：1）通过构建嵌合I/LFABP，确定α I螺旋和α II螺旋在脂肪酸向膜的碰撞转移中的重要性; 2）采用定点突变技术，阐明特定带电和疏水残基在FA向膜的碰撞转移中的作用。 这种方法的脂肪酸转运功能的脂肪酸结合蛋白应提供分子水平的细节的具体结构元素，导致独特的运输性能的IFABP与LFABP。 具体而言，对于所有点突变和嵌合蛋白工程，我们将分析的速率和机制的脂肪酸转移到模型膜使用荧光共振能量转移试验，和膜的蛋白质的相互作用，使用几个互补的生物化学和生物物理技术。 由于LFABP和IFABP都在小肠上皮细胞中高度表达，因此对其作用机制的理解将使膳食脂质同化的有效营养和/或药理学操作成为可能。

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.