ATP BINDING SITE PHOTOAFFINITY PROBES FOR F1-ATPASE

本研究的长期目标是阐明心绞痛的发病机制。 多亚单位F1-ATPase，最重要但结构复杂的酶之一 酶，无处不在地负责创造正常的大部分ATP 和病态细胞。它由两部分组成，其中一部分是膜 结合并作为跨膜质子通道(F0)；另一部分 催化ADP+PI(F1)合成三磷酸腺苷。F1要求 用于至少2个催化核苷酸的催化协同参与 不同(β)亚基上的结合位点。新设计的荧光灯 光亲和核苷酸类似探针(BzAF和BzAE，后者 具有重碘原子)，它仅在 它的3个催化位置，表明当这些位置顺序 当被占领时，F1发生了催化前构象变化。这会导致 结合底物的每个位置的亲和力的协调变化 配基，与当前被接受的“结合”的过程辅音 改变/交替位置“机制。与我们先前设计的 并成功地使用了二苯甲酮衍生的核苷酸集合 光亲和标记(BzAT(D)p)，要解决的问题 澄清如下：1)特定的Beta亚基存在哪些差异 与核苷酸结合的多肽结构域和氨基酸残基。 一种以上摩尔的底物类似物配体共价连接到 每个摩尔酶的催化位置？2)是否存在跨膜 质子梯度有助于F1的构象变化？3)什么是 协同作用的催化核苷酸之间的距离 F1全酶上的结合位点？4)具体的拓扑结构是什么 催化(β亚基)核苷酸位点(S)在F1中的定位 X射线结晶学推断的多亚基全酶？吃什么？ 其他生物催化所需的构象调整 多核苷酸利用酶，如肌腺苷酸激酶(ADK)作为 通过我们新的荧光、核苷酸类似光亲和探针？ 解决这些问题的实验方法应包括：1)交联 控制不同种类的F1(细菌和线粒体) 用我们的BZ-光亲和探针库标记化学计量 (BzAT(D，M)P，BzAF和BzAE)；2)F1的高分辨蛋白质测序 与一摩尔以上的BZ探针共价标记；3)稳定 质子梯度诱导的态荧光研究 结合位点占位引起的F1基因的构象变化 重组膜泡囊系统；4)荧光BzAF和 BzAE(或ADN-Tb3+)探针作为双标记F1 to的光谱对 表征催化剂之间的相互作用和距离的测量 通过Forster能量转移；5)使用四碘BzAE作为一种 X射线结构中核苷酸结合的本征拓扑标记 BZAE标记的F1晶体。

The long term goal of this research is to clarify the mechanism of the multisubunit F1-ATPase, one of the most important yet structurally complex enzymes, ubiquitously responsible for creation most of the ATP of normal and diseased cells. It consists of two parts, one of which is membrane bound and serves as a transmembrane proton channel (F0); the other part performs the catalytic synthesis of ATP from ADP + Pi(F1). The F1 requires for catalysis cooperative participation of at least 2 catalytic nucleotide binding sites on different (beta) subunits. Newly designed fluorescent photoaffinity nucleotide analog probes (BzAF and BzAE, the latter possessing heavy iodine atoms), which covalently crosslink to F1 only at its 3 catalytic sites, indicate that when these sites are sequentially occupied, a pre-catalytic conformational change in F1 occurs. This causes a coordinated change in the affinity of each site for the bound substrate ligands, a process consonant with the currently accepted "binding change/alternating site" mechanism. Together with our previously designed and successfully employed set of benzophenone-derivatized nucleotide photoaffinity labels (BzAT(D)p), the questions to be addressed for clarification are: 1) What differences exist in the specific Beta subunit peptide domains and amino acid residues that bind nucleotide when one vs. more-than-one mol of substrate analog ligand is covalently linked to a catalytic site per mol enzyme? 2) Does the existence of a transmembrane proton gradient contribute to a conformational change in F1? 3) What is the distance between the cooperatively interacting catalytic nucleotide binding sites on the F1 holoenzyme? 4) What is the specific topological location of the catalytic (beta subunit) nucleotide site(s) within the F1 multisubunit holoenzyme as deduced via X-ray crystallography? 5) What ate the conformational adjustments required for catalysis by other multinucleotide-utilizing enzymes, such as muscle adenylate kinase (ADK) as disclosed via our new fluorescent, nucleotide analog photoaffinity probes? Experimental methods to solve these problems shall include: 1) Crosslinking of various species of F1 (bacterial as well as mitochondrial) to controlled labelling stoichiometries with our arsenal of Bz-photoaffinity probes (BzAT(D,M)P,BzAF and BzAE;2) High resolution protein sequencing of F1 covalently labelled with on vs. more-than-one mol of Bz probe; 3) Steady state fluorescence studies of proton gradient-induced, vs. sequential binding site occupancy-induced, conformational changes in F1 in reconsituted membrane vesicle systems; 4) Use of the florescent BzAF and BzAE (or AdN-Tb3+) probes as spectroscopic pairs with bi-labelled F1 to characterize interactions and measurements of distances between catalytic sites, via Forster energy transfer; 5) Use of the tetraiodinated BzAE as an intrinsic topological marker of nucleotide binding in the x-ray structure of the BzAE labelled F1 crystal.