ATP BINDING SITE PHOTOAFFINITY PROBES FOR F1-ATPASE

本研究的长期目标是阐明 多亚基F1-ATP酶，其中一个最重要的，但结构复杂 酶，普遍负责创造大多数正常的ATP， 和病变细胞。 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 由ADP + Pi催化合成ATP（F1）。 F1需要 对于催化，至少2个催化核苷酸协同参与 不同（β）亚基上的结合位点。 新设计的荧光 光亲和核苷酸类似物探针（BzAF和BzAE，后者 具有重碘原子），其仅在20 ℃下与F1共价交联。 它的3个催化位点，表明当这些位点依次 占据时，F1发生催化前构象变化。 这导致 每个位点对结合底物的亲和力的协调变化 配体，一个与目前公认的“结合”相一致的过程， 改变/交替位点”机制。 加上我们以前设计的 并成功地采用了二苯甲酮衍生的核苷酸 光亲和标记（BzAT（D）p），要解决的问题， 澄清是：1）在特定的β亚基中存在什么差异 肽结构域和氨基酸残基结合核苷酸时，一个与 超过1摩尔的底物类似物配体共价连接到 每摩尔酶的催化位点？2)跨膜蛋白的存在 质子梯度有助于F1的构象变化？ 3)是什么 协同相互作用的催化核苷酸之间的距离 F1全酶上的结合位点 4)具体的拓扑是什么 F1中催化（β亚基）核苷酸位点的位置 多亚基全酶通过X射线晶体学推断？ 5)什么吃 其他分子催化所需的构象调整 多核苷酸利用酶，如肌肉腺苷酸激酶（ADK）， 通过我们新的荧光，核苷酸类似物光亲和探针披露？ 解决这些问题的实验方法应包括：1）交联 不同种属的F1（细菌和线粒体）与对照 用我们的Bz光亲和探针标记化学计量 （BzAT（D，M）P、BzAF和BzAE;2）F1的高分辨率蛋白质测序 共价标记的Bz探针; 3）稳定 质子梯度诱导与顺序诱导的状态荧光研究 结合位点占据诱导的F1构象变化， 重构的膜囊泡系统; 4）使用荧光素BzAF和 BzAE（或AdN-Tb 3+）探针作为光谱对，具有双标记的F1至 表征催化剂之间的相互作用和距离测量 5）使用四碘化的BzAE作为一种催化剂， X射线结构中核苷酸结合的内在拓扑标记 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.