Calorimetric and fluorescence studies of protein propert

CLP分子伴侣对底物的识别依赖于与由特定多肽序列组成的基序的相互作用。我们研究了在三磷酸腺苷和镁(II)存在下，含短基序的多肽在pH 7.5的条件下与大肠杆菌依赖于三磷酸腺苷的ClpAP酶的伴侣成分ClpA的结合。用等温滴定热法(ITC)测定了AANDENYALAA多肽的结合，AANDENYALAA多肽对应于体内异常新生多肽C末端的SsrA降解基序。每个ClpA六聚体结合一个SsrA多肽，结合常数为5E+6 1/M，结合结合的N端丹化SsrA多肽的荧光强度与化学计量比相同(结合常数为1E+7 1/M)。在6 C位用ATP取代ATPGammaS也得到了类似的结果，另外两个来自噬菌体P1 REPA蛋白和E.ColiHEMA蛋白的多肽是SsrA结合的竞争性抑制剂，与ClpA六聚体结合的表观结合常数为&gt；3 E+7 1/M。由于ClpA六聚体一次只能展开和转运一种蛋白质，限制最初结合的多肽数量将有助于避免底物的非生产性结合和部分加工蛋白质的聚集。 钙结合蛋白Calmyrin被认为是一种重要的信号蛋白。该蛋白包含四个EF-Hand基序，其中两个以高亲和力结合Ca(II)。Calmyrin在人体多种组织中表达，并与广泛的蛋白质相互作用，但这种单一蛋白质用来向如此多的靶点传递钙信号的机制尚不清楚。一种可能的钙(II)诱导机制包括暴露与蛋白质的α-螺旋结合的疏水口袋。第二种可能的机制涉及钙(II)-肉豆蔻酸基开关，导致钙(II)结合蛋白锚定在细胞内或质膜上。钙化肌蛋白已被证明在其N-末端是肉豆蔻酰化的，但到目前为止还没有发现依赖于Ca(II)的细胞膜转位。第三种机制可能与钙(II)依赖的齐聚反应有关。为了确定寡聚在Ca(II)信号转导中的可能作用，我们对重组Calmyrin的寡聚进行了表征，并鉴定了具有生物活性的calmyrin形式。非还原的SDS/PAGE分析表明，在体外，apo和Ca(II)结合的钙麦芽蛋白发生寡聚，形成稳定的分子间二硫键。还原条件下的超速离心实验表明，脱脂蛋白存在21.9 kDa的单体和43.8 kDa的二聚体的平衡，6℃时的二聚常数为1.8E+31/M，这一结果被脱脂蛋白和钙离子结合的脱脂蛋白凝胶过滤证实。重要的是，单体和二聚体都发生了显著的构象变化，以响应结合的钙(II)。然而，当在非还原条件下用Western blotting分析细胞提取物时，在人的血小板和淋巴细胞以及大鼠的脑中只检测到单体钙调蛋白。此外，与重组钙化蛋白相比，无论Ca(II)浓度如何，交联实验都无法检测到任何二聚体形式的钙化蛋白。综上所述，我们目前的数据表明，虽然钙调蛋白在体外形成稳定的共价二聚体，但在体内似乎是作为单体发挥作用。

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.