P450BM3--STRUCTURE AND FUNCTION

P450被认为是严格的排毒/激素合成 哺乳动物中的酶；然而，随着对P450的鉴定， 哺乳动物、植物、真菌和细菌，人们已经认识到它的作用 P450的多样性远远超出了人们之前的猜测。与 P450cam和另外两个P450在我们的 实验室，P450BM-P和P450terp，我们已经能够确定 结构元素是高度保守的，哪些元素较少 保守的。这些P450‘S在序列上具有极高的变异性 (不到10%的同一性)；然而，总体上有一个三维的 结构，即与P450cam，P450terp相似的结构褶皱， 和P450BM-P。 这个项目所围绕的假设是，存在一个 P450结构折叠，因此，一个特定的折叠途径，它 描述了这个蛋白质超家族的特征。对这一点的理解 结构折叠是修饰P450酶活性的关键 用于生物修复和药物合成。因此，我们的目标是 为了建立折叠、展开、血红素重组的条件 为了能够修饰底物的特异性和氧化还原伙伴的结合。 具体地说，为了检验我们的假设，我们提出了以下目标； A.评估P450BM-P中的折叠/去折叠途径 I.为变性和随后的变性创造条件 复性 二、用血红素及其衍生物制备P$%)BM-P 底物/氧结合袋和血红素环境。 B.识别那些允许正确折叠的结构元素， 用于底物结合，以及用于氧化还原伙伴结合 I.利用合成的寡核苷酸合成P450BM-P 二、系统地消除结构元素，构建一个“最低限度” P450 三、取代其他P450 S的结构元素构建 具有杂合活性的杂合蛋白 C.确定P450形式的三维结构 最感兴趣/最有用处 I.评价与花生四烯酸结合的P450BM-P的结构 或二十碳五烯酸 二、结晶并确定这些杂交/突变的P450的结构 这可能有助于揭示P450折叠途径的最小结构。 圆二色谱将被用来评估 这些蛋白质在折叠和去折叠过程中的三级结构，而 磁性圆二色谱将被用来研究 亚铁血红素的配基状态。

p450S have been thought of as strictly detoxification/hormone synthesizing enzymes in mammals; however, with the identification of more than P450s in mammals, plants, fungi, and bacteria, it has been realized that the role of P450s is much more diverse than had been previously suspected. With the elucidation of the structure of P450cam and of two additional P450s in our laboratory, P450BM-P and P450terp, we have been able to determine which structural elements are highly conserved and which elements are less conserved. These P450's have an extremely high variability in sequence (less than 10% identity); however, there is an overall three dimensional structure, i.e. a structural fold which is similar in P450cam, P450terp, and P450BM-P. The hypothesis around which this project has coalesced is that there is a P450 structural fold, and hence, a specific folding pathway which characterized this superfamily of proteins. The understanding of this structural fold is critical in the efforts to modify P450 enzyme activities to be used in bioremediation and drug synthesis. Therefore, our aims are to establish conditions for folding, unfolding, an heme reconstitution in order to be able to modify substrate specificity and redox partner binding. Specifically, to test our hypothesis we have set forth the following aims; A. Evaluating the folding/unfolding pathway in P450BM-P i. establishment of conditions for denaturation and subsequent renaturation ii. preparation of P$%)BM-P with heme and heme derivatives to probe the substrate/oxygen binding pocket and heme environment. B. Identifying those structural elements which allow for correct folding, for substrate binding, and for redox-partner binding i. synthesis of P450BM-P using synthetic oligonucleotides ii. systematic elimination of structural elements to construct a "minimal" P450 iii. substitution of structural elements from other p450 s to construct hybrid proteins with hybrid activities C. Determining the three dimensional structures of those p450 forms which are of greatest interest/utility i. evaluate the structure of P450BM-P bound to the substrates arachidonic or eicosapenteneoic acid ii. crystallize and determine the structure of those hybrid/mutant P450s which might shed light on the minimal structure of a P450 folding pathway. Circular dichroism spectroscopy will be used to assess the changes in tertiary structure during folding and unfolding of these proteins, while magnetic circular dichroism spectroscopy will be used to examine changes in the ligand state of the heme.