Calorimetric and fluorescence studies of protein propert

Clp分子伴侣的底物识别依赖于与由特定肽序列组成的基序的相互作用。我们研究了带有短基序的肽与ClpA的结合，ClpA是E.在pH 7.5的ATP γ S和Mg（II）存在下，通过等温滴定量热法（ITC）使用肽AANDENYALAA测量结合，所述肽AANDENYALAA对应于在体内异常新生多肽的C末端发现的SsrA降解基序。每个ClpA的六聚体环结合一个SsrA肽，结合常数为5 E+6 1/M。还通过N-末端丹磺酰化SsrA肽的荧光变化来测定结合，所述肽以每个ClpA六聚体一个的相同化学计量结合（缔合常数为1 E+7 1/M）。当在6 ℃下用ATP代替ATP γ S时，获得了类似的结果。另外两个肽来源于噬菌体P1 RepA蛋白和E. coli HemA蛋白是SsrA的竞争性抑制剂，与ClpA六聚体的结合常数> 3E +71/M。DNS-SsrA仅以略微降低的亲和力与缺失N端结构域或C端核苷酸结合结构域的ClpA缺失突变体结合，表明SsrA的结合位点位于N端核苷酸结合结构域内。因为一次只有一种蛋白质可以被ClpA六聚体解折叠和易位，所以限制最初结合的肽的数量将有助于避免底物的非生产性结合和部分加工的蛋白质的聚集。 钙（II）结合蛋白calmyrin被认为是一种重要的信号蛋白。这种蛋白质含有四个EF-手的动机，其中两个结合Ca（II）具有高亲和力。钙蛋白在多种人体组织中表达，并与广谱蛋白质相互作用，但这种单一蛋白质将Ca（II）信号传递到多种靶点的机制仍然未知。一种可能的Ca（II）诱导机制涉及暴露结合蛋白质的α-螺旋的疏水口袋。第二种潜在机制涉及Ca（II）-肉豆蔻酰开关，其导致Ca（II）结合蛋白锚定到细胞内或质膜。钙蛋白已被证明是肉豆蔻酰化在其N-末端，但迄今为止，钙（II）依赖性易位到细胞内膜尚未得到证实。第三种机制可能与Ca（II）依赖性寡聚化有关。要建立一个可能的作用，寡聚化的钙（II）信号转导，我们的特点是寡聚化的重组calmyrin和确定生物活性calmyrin形式。非还原SDS/PAGE显示，在体外脱辅基和钙（II）结合钙蛋白寡聚化，形成稳定的分子间二硫键。在还原条件下的超离心表明脱辅基钙蛋白以21.9kDa单体和43.8kDa二聚体的平衡存在，在6 ℃下二聚常数为1.8E +31/M。这一结果证实了载脂蛋白和钙（II）结合钙蛋白的凝胶过滤。重要的是，单体和二聚体都经历了显着的构象变化，响应于结合的Ca（II）。然而，当在非还原条件下通过Western印迹法分析细胞提取物时，在人血小板和淋巴细胞以及大鼠脑中仅检测到单体钙蛋白。此外，与重组钙蛋白相反，交联实验无法检测到任何钙蛋白的二聚体物质，无论Ca（II）浓度如何。总之，我们目前的数据表明，虽然钙蛋白在体外形成稳定的共价二聚体，但它似乎在体内起单体的作用。

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of E. coli in the presence of ATPgammaS and Mg(II) at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C-terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexameric ring of ClpA with an association constant of 5 E+6 1/M. Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (association constant of 1 E+7 1/M). Similar results were obtained when ATP was substituted for ATPgammaS at 6 C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamer with the apparent association constant >3 E+7 1/M. DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound will help to avoid non-productive binding of substrates and aggregation of partially processed proteins. The Ca(II)-binding protein calmyrin is believed to be an important signaling protein. This protein contains four EF-hand motives, two of which bind Ca(II) with high affinities. Calmyrin is expressed in a variety of human tissues and interacts with a broad spectrum of proteins, yet the mechanisms used by this single protein to transmit Ca(II) signals to such a variety of targets remains unknown. One possible Ca(II)-induced mechanism involves exposure of hydrophobic pockets that bind to alpha-helices of a protein. Second potential mechanism involves a Ca(II)-myristoyl switch that leads to the anchoring of Ca(II)-binding proteins to intracellular or plasma membranes. Calmyrin has been shown to be myristoylated at its N-terminus, but thus far Ca(II)-dependent translocation to intracellular membranes has not been demonstrated. A third mechanism may be related to a Ca(II) dependent oligomerization. To establish a possible role of oligomerization in Ca(II)-signal transduction, we characterized oligomerization of the recombinant calmyrin and identified biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(II)-bound calmyrin oligomerizes, forming stable intermolecular disulfide bridges. Ultracentrifugation under reducing conditions indicated that apo-calmyrin exists in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer, with dimerization constant of 1.8 E+3 1/M at 6 C. This result was confirmed by gel filtration of apo- and Ca(II)-bound calmyrin. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(II). However, when cell extracts were analyzed under non-reducing conditions by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brains. Moreover, in contrast to the recombinant calmyrin, crosslinking experiments were unable to detect any dimeric species of calmyrin regardless of Ca(II) concentrations. In summary, our current data indicate that although calmyrin forms stable covalent dimmers in vitro, it appears to function as a monomer in vivo.