Protein Folding and Stability, Macromolecular Interactions, and Ligand Binding

酶I：GP、AP 氚标记蛋白质是结构研究中常用的一种技术。我们想要探索氚标记是如何影响酶的稳定性和活性的。作为本研究的模型蛋白，我们选择了大肠杆菌一个重要的新的氮信号通路的第一个酶，即I-NTR酶(EI-NTR)。EI-NTR的His356残基在EI-NTR与PEP/Mg(II)的自催化反应中被磷酸化，这种高能的磷酸被转移到NPR的His16，这是一种含有90个氨基酸的小载体蛋白。我们用等温滴定量热法、分析超速离心法和光散射法获得了EI-NTR及其单体氨基末端结构域EIN-NTR与受体蛋白NPR相互作用的热力学参数。用差示扫描量热法比较了EI-NTR与其氚标记的EI-NTR的稳定性，发现DH酶的稳定性受到影响，其Tm降低了4℃。我们继续从热力学的角度研究氢化的EI-NTR与其配体的相互作用，以了解这种酶的活性的哪些方面会受到氚标记的影响。 马传染性贫血病毒(EIAV)基质蛋白的寡聚体状态。(GP，新界) 马传染性贫血病毒(EIAV)基质蛋白结构域(MA)在病毒成熟过程中将病毒前体多肽靶向于感染细胞膜的胞浆侧。尽管序列同源性很低，但各种逆转录病毒的MA结构域在大小上相似(14-15 kDa)，并共享一个由5个螺旋组成的高度保守的三级结构。有趣的是，HIV-1 MA结晶为三聚体，而EIAV MA结晶为非对称二聚体。然而，荧光研究表明，EIAV MA以两到三个亚基的多聚体形式存在。我们进行了一系列分析超速离心法来表征EIAV MA在溶液中的寡聚形式。我们的结果与微摩尔浓度下EIAV MA的初级单体-三聚体平衡模型是一致的。 ClpA低聚体的构象动力学。(GP、MRM) ClpA是一种来自E.Coli的Hsp100/CLP伴侣蛋白，它形成一个六聚体亚基环，在依赖于ATP的过程中解开天然蛋白质，并将未折叠的多肽递送到ClpP蛋白酶进行降解。三磷酸腺苷驱动ClpA展开酶活性的确切机制很重要，但还没有很好地阐明。以前的ClpA六聚体与SsrA靶肽的量热滴定表明，肽结合可能会引发ClpA六聚体内的构象变化。这一观察结果得到了分析性超速离心研究的证实，该研究表明，SsrA肽结合引起的ClpA六聚体构象变化可以通过沉降系数分布来反映。 我们继续使用这种方法来进一步了解单个ClpA域的作用。ClpA六聚体的每个亚基都有两个ATP结合域(d1和d2)和位于中央通道末端的N结构域。使用分别缺少N-末端结构域和ClpP对接D2结构域的ClpA-Deltan和ClpA-ND1突变体，我们能够建立起与ClpA六角体结合底物相关的大部分构象变化是由环状D2结构域负责，而不是由可能的柔性N结构域负责。基于这些结果的ClpA底物处理机制的模型进一步得到了ClpA缺失突变体对ATP酶活性刺激的数据的支持。在这些实验中，通过分析超速离心法表现出较大构象变化的蛋白质，其三磷酸腺苷酶活性也被刺激，反之亦然。 封端蛋白-CARMIL相互作用研究。(GP、TU、JAH) Capping蛋白(CP)是一种高度保守的肌动蛋白结合蛋白，对正常的肌动蛋白动力学是必不可少的，它是许多细胞过程的重要组成部分，包括免疫反应和癌细胞增殖。CP结合到肌动蛋白细丝的带刺末端，阻止肌动蛋白单体的结合和解离。一个潜在的CP调节因子是CARMIL，它可能作为一种强有力的CP拮抗剂，抑制CP与肌动蛋白细丝的相互作用。 CARMIL蛋白的两个区域(CAH3-a和CAH3-b)先前被认为是与CP相互作用的关键。利用含有CAH3-a和CAH3-b区序列的CARMIL克隆片段，用等温滴定量热法(ITC)研究了CARMIL与小鼠冠状蛋白(MCP)的相互作用。将ITC获得的结果与肌动蛋白聚合抑制实验的结果进行比较，以确定有助于结合亲和力的CARMIL区域和解帽活性所必需的区域。我们继续使用这一有效的策略通过研究ITC滴定中的几个单点MCP突变来定位MCP与CARMIL结合的活性位点。将这些实验的结果与使用MCP的固有色氨酸荧光的荧光滴定进行了比较。

Enzyme I: GP, AP Deuterium labeling of proteins is a commonly used technique in structural studies. We would like to explore how deuterium labeling affects the stability and activity of enzymes. As a model protein in ths study we have selected the first enzyme of an important new nitrogen signal pathway of E. Coli, the enzyme I-Ntr (EI-Ntr). The His356 residue of EI-Ntr is phosphorylated in the autocatalytic reaction of EI-Ntr with PEP/Mg(II) and this high energy phosphate is transferred to His16 of NPr, a small carrier protein containing 90 amino acids. We have obtained thermodynamic parameters for dimerization of EI-Ntr and for interactions of EI-Ntr and its monomeric amino terminal domain EIN-Ntr with the acceptor protein NPr using isothermal titration calorimetry, analytical ultracentrifugation and light scattering methods. Comparing the stability of EI-Ntr with its deuterium labeled counterpart we observed that the stability of deuterated enzyme is compromised and the Tm of the deuterated EI-Ntr is lowered by 4 degrees K as shown by the differential scanning calorimetry. We continue to thermodynamically characterize interactions of the deuterated EI-Ntr with its ligands to understand what aspects of the activity of this enzyme are affected by deuterium labeling. Oligomeric state of the equine infectious anemia virus (EIAV) matrix protein. (GP, NT) Equine infectious anemia virus (EIAV) matrix protein domain (MA) is targeting the viral precursor polypeptide to the cytosolic side of the infected cell membrane during the virus maturation. Despite poor sequence homology, MA domains from various retroviruses are similar in size (14-15 kDa) and share a highly conserved tertiary structure composed of five helices. Interestingly, HIV-1 MA crystallized as a trimer while EIAV MA was crystallized in a nonsymmetric dimeric unit. Nevertheless, fluorescence studies suggest that EIAV MA exists as a multimer of two to three subunits. We carried out a series of analytical ultracentrifugation measurements to characterize the oligomeric form of EIAV MA in solution. Our results are consistent with a primary monomer-trimer equilibrium model for EIAV MA at micromolar concentrations. Conformational dynamics of the ClpA oligomers. (GP, MRM) ClpA, a Hsp100/Clp chaperone from E. Coli, forms a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. The precise mechanism of ATP driven unfoldase activity of ClpA is important but not well elucidated. Previous calorimetric titrations of ClpA hexamer with SsrA target peptide have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. This observations were confirmed by analytical ultracentrifugation studies indicating that ClpA hexamer conformational changes induced by SsrA peptide binding are reflected by sedimentation coefficient distributions. We continue to use this methodology to gain further insight into the role of individual ClpA domains. Each subunit of the ClpA hexamer has two ATP binding domains (D1 and D2) and the N domain located at the end of the central channel. Using the ClpA-deltaN and the ClpA-ND1 mutants, lacking the N-terminal domain and the ClpP docking D2 domain, respectively, we were able to establish that the ring-forming D2 domain, rather then the presumably flexible N domain, is responsible for most of the conformational changes associated with substrate binding by the ClpA hexamer. Models of the ClpA substrate processing mechanism based on those results were further supported by the data of the ATP-ase activity stimulation obtained with ClpA deletion mutants. In those experiments proteins showing large conformational changes by analytical ultracentrifugation were also stimulated in their ATP-ase activity and vice versa. Capping Protein - CARMIL Interaction Studies. (GP, TU, JAH) Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics which is an important part of many cellular processes, including immunological responses and cancer cell proliferation. 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. Two regions of the CARMIL protein (CAH3-a and CAH3-b) were previously identified as crucial for interactions with CP. Cloned fragments of CARMIL with sequences encompassing both the CAH3-a and CAH3-b regions, with several single point mutations with either of them, were used to study CARMIL interaction with mouse Capping Protein (mCP) by Isothermal Titration Calorimetry (ITC). Results obtained by ITC were compared with those from an actin polymerization inhibition assay allowing for the identification of CARMIL regions contributing to binding affinity and those necessary for uncapping activity. We continue to use this effective strategy to locate mCP active sites for CARMIL binding by studying several single point mCP mutants in ITC titrations. Results of those experiments are compared with fluorescence titrations where the intrinsic Tryptophane fluorescence of mCP is used.