Protein Folding and Stability, Macromolecular Interactio

ClpA六聚体的构象动力学。(GP、AG、MRM） ClpA是来自E.大肠杆菌中，是亚基的六聚环，其在ATP依赖性过程中展开天然蛋白质并将展开的多肽递送至ClpP蛋白酶进行降解。ClpA对底物的识别依赖于与特定标签的相互作用，例如附加到蛋白质C-末端的11个氨基酸的SsrA序列，该蛋白质在核糖体上被限制性地停滞。为了深入了解ClpA六聚体在肽结合后的整体构象变化，我们使用沉降速度测量来确定ClpA在不存在和存在10倍过量SsrA的情况下在不同温度下的沉降系数（S）分布。先前用SsrA对ClpA六聚体进行的量热滴定表明，肽结合可能引发ClpA六聚体内的构象变化。分析超离心研究表明，肽结合的ClpA表现出一个显着更窄的分布比ClpA沉降在肽的情况下的沉降系数。对于野生型ClpA和缺乏N-末端调节结构域的缺失突变体（ClpADN）获得了类似的结果。由SsrA肽结合诱导的ClpA六聚体中的构象变化，如由S分布所反映的，是温度依赖性的。总体而言，沉降速度结果表明，底物肽结合稳定了ClpA六聚体的独特构象。 为了进一步研究ClpA六聚体的构象动力学和肽底物易位的动力学，我们制备了ClpA的双半胱氨酸突变体。该突变体将用含有长荧光寿命供体的供体-受体染料对标记。初步研究证实，能量转移测量与金属配体络合物为基础的荧光团作为供体可能会揭示信息的微秒动力学的CpA蛋白。 枯草芽孢杆菌ClpP蛋白酶的寡聚化和蛋白水解活性。(GP、AG、MRM、DS） ClpXP和ClpAP复合物的蛋白水解组分，ClpP蛋白酶，在从真细菌到高等真核生物的线粒体的不同物种中是非常保守的。该系统已在革兰氏阴性菌E.大肠杆菌中，但在革兰氏阳性细菌如枯草芽孢杆菌中研究ClpP的兴趣越来越大。最近，已经表明ClpP是革兰氏阳性细菌中一类新抗生素的靶标。还有一些证据表明ClpP可能以其单体形式存在于枯草芽孢杆菌中。我们克隆了野生型和组氨酸标记的枯草芽孢杆菌ClpP基因，并在大肠杆菌中表达。大肠杆菌表达，并纯化蛋白。我们的数据表明，克隆的枯草芽孢杆菌ClpP与E. Coli ClpX蛋白。凝胶过滤实验表明枯草芽孢杆菌ClpP在体外形成十四聚体复合物。枯草芽孢杆菌ClpP的分析超离心研究正在进行中。 人过氧化物氧还蛋白I（hPrxI）的氧化依赖性寡聚化及其与人硫氧还蛋白（hSrx）的相互作用。(GP、AG、DYL、SGR） 人Peroxiredoxin I属于一个庞大而多样的抗氧化酶家族，也调节细胞信号传导途径、凋亡和分化。过氧化物酶（Prxs）可以通过磷酸化和氧化还原状态的变化来调节。寡聚化被认为在这种调节中起关键作用。大多数2-Cys Prx在寡聚状态下表现出显著的变化，形成具有特征性甜甜圈形状的二聚体或十聚体。 目前，我们正在研究hPrx I的还原，氧化和过氧化（Cys-SO2H）形式的这种酶和这些复合物与人硫氧还蛋白（hSrx）的相互作用的氧化还原依赖性寡聚化。沉降速率实验表明，hPrx氧化还原依赖的寡聚反应与文献报道的其他Prx有显著不同。氧化的hPrx是专性同二聚体，并且该酶的还原形式处于浓度依赖性单体-二聚体-十聚体平衡。过氧化形式的hPrx也形成十聚体，并且该过程的浓度依赖性高于该酶的还原形式。将使用多信号沉降速度方法来获得关于hSrx与溶液中不同低聚形式的hPrx的相互作用的化学计量的信息。 加帽蛋白-CARMIL相互作用研究。(GP、AG、TU、JAH） 加帽蛋白（CP）是一种高度保守的肌动蛋白结合蛋白，是正常肌动蛋白动力学所必需的。CP结合到肌动蛋白丝的倒刺末端，阻断肌动蛋白单体的结合和解离。CARMIL是CP的一个潜在调节因子，它可能作为一种有效的CP拮抗剂，抑制CP与肌动蛋白丝的相互作用。 正在通过等温滴定量热法研究小鼠加帽蛋白（mCP）异二聚体（a，B）与小鼠CARMIL不同片段的相互作用，以鉴定结合位点。通过在不同温度下进行这些实验，将获得热容信息。

Conformational dynamics of the ClpA Hexamer. (GP, AG, MRM) ClpA, a Hsp100/Clp chaperone from E. Coli, is a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. Substrate recognition by ClpA is dependent on interactions with specific tags, such as the 11-amino acid SsrA sequence appended to the C-termini of proteins translationally stalled at ribosomes. To gain insight into the overall conformational changes of the ClpA hexamer upon peptide binding, we have used sedimentation velocity measurements to determine sedimentation coefficient (S) distributions of ClpA in the absence and presence of 10-fold excess of SsrA at different temperatures. Previous calorimetric titrations of ClpA hexamer with SsrA have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. Analytical ultracentrifugation studies indicate that peptide-bound ClpA exhibits a strikingly more narrow distribution of sedimentation coefficients than does ClpA sedimenting in the absence of the peptide. Similar results are obtained for the wild-type ClpA and for a deletion mutant lacking the N-terminal regulatory domain (ClpADN). The conformational change in ClpA hexamer induced by SsrA peptide binding, as reflected by the S distribution, is temperature dependent. Overall, sedimentation velocity results indicate that substrate peptide binding stabilizes a unique conformation of the ClpA hexamer. To further study conformational dynamics of the ClpA hexamer and the dynamics of peptide substrate translocation, we are preparing a double cysteine mutant of ClpA. This mutant will be labeled with a donor-acceptor dye pair containing a long fluorescence lifetime donor. Preliminary studies confirmed that energy transfer measurements with metal-ligand complex based fluorophores as donors may reveal information on microsecond dynamics of CpA protein. Oligomerization and proteolytic activity of the ClpP protease from Bacillus subtilis. (GP, AG, MRM, DS) The proteolytic component of the ClpXP and ClpAP complexes, the ClpP protease, is well conserved among different species from eubacteria to the mitochondria of higher eukaryotes. This system has been well studied in the gram-negative bacteria E. Coli, but there is a growing interest in studying ClpP in gram positive bacteria such as Bacillus subtilis. Recently, it has been shown that ClpP is a target of a new class of antibiotic in gram positive bacteria. There is also some evidence suggesting that ClpP may be present in its monomeric form in Bacillus subtilis. We cloned both wilde type and histidine-tagged Bacillus subtilis ClpP gene, expressed this gene in the E. Coli, and purified the proteins. Our data show that cloned Bacillus subtilis ClpP forms functional complexes with E. Coli ClpX proteins. Gel filtration experiments suggest that Bacillus subtilis ClpP forms tetradecamer complexes in vitro. Analytical ultracentrifugation studies of the Bacillus subtilis ClpP are underway. Oxidation dependent oligomerization of the human Peroxiredoxin I (hPrxI) and its interactions with human Sulfiredoxin (hSrx). (GP, AG, DYL, SGR) Human Peroxiredoxin I belongs to a large and diverse family of antioxidant enzymes that also regulate cell signaling pathways, apoptosis and differentiation. Peroxiredoxins (Prxs) can be regulated by changes to phosphorylation and redox state. Oligomerization is believed to play a key role in this regulation. Most of 2-Cys Prxs demonstrate dramatic changes in oligomeric state, forming either dimers or decamers with the characteristic doughnut shape. Currently we are studying redox-dependent oligomerization of hPrx I for both reduced, oxidized and overoxidized (Cys-SO2H) forms of this enzyme and interactions of those complexes with the human Sulfiredoxin (hSrx). Sedimentation velocity experiment show, that hPrx redox-dependent oligomerization is strikingly different from other Prxs, reported in literature. The oxidized hPrx is an obligate homodimer and the reduced form of this enzyme is in concentration dependent monomer-dimer-decamer equilibrium. The overoxidized form of hPrx also forms decamers, and the concentration dependence of this process in higher than that of the reduced form of this enzyme. Multisignal sedimentation velocity method will be used to obtain information on the stoichiometry for interactions of hSrx with different oligomeric forms of hPrx in solution. Capping Protein - CARMIL Interaction Studies. (GP, AG, TU, JAH) Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics. CP binds to the barbed end of the actin filament blocking both association and dissociation of actin monomers. One potential regulator of CP is CARMIL, that might act as potent CP antagonist and inhibit CP interaction with actin filaments. Interactions of Mouse Capping Protein (mCP) heterodimer (a, b) with different fragments of mouse CARMIL are being studied by isothermal titration calorimetry to identify binding sites. By conducting these experiments at different temperatures, heat capacity information will be obtained.