Calorimetric and fluorescence studies of protein propert

底物多肽与ClpA六聚体的相互作用。(GP、AG、MRM、SKS、JAR) ClpA是一种依赖于ATP的伴侣，它形成一个六聚体环，在体外单独具有去折叠酶活性。其中两个环位于形成ClpP十四聚体的ClpAP蛋白酶的侧翼。在这个复合体中，ClpA通过结合、展开底物和将底物转移到蛋白水解室来帮助蛋白水解室。用已知11个氨基酸底物ANDENYALAA(SsrA)和α-SsrA的等温滴定热法(ITC)和丹磺化SsrA多肽的荧光滴定(DNS-SsrA)测定了ClpA与多肽相互作用的热力学参数。已发现Dns-SsrA被ClpA的其他底物竞争性取代。在荧光研究中，含有已知底物ClpA和ClpX不同部分序列的多肽被用于与Dns-SsrA竞争。这项技术允许识别负责ClpA识别的特定基序。荧光方法也被用来研究接头蛋白，如CLP，对ClpA-多肽相互作用的影响。来自不同底物的肽序列与DNS-SsrA竞争的事实表明，它们占据或重叠了ClpA六聚体环的同一结合部位。 葡萄糖/半乳糖结合蛋白的构象稳定性和结构域偶联。(GP、AG、SA、MS、MR) 来自大肠杆菌的D-葡萄糖/D-半乳糖结合蛋白(GGBP)是一种周质蛋白，是一种高亲和力的受体，对这两种糖具有主动转运和趋化作用。糖的紧密结合是通过与配体形成氢键实现的，结合部位位于GGBP两个结构域之间的缝隙中。葡萄糖和半乳糖结合诱导GGBP蛋白的两个结构域之间的铰链运动。由于GGBP可以通过监测诱导的蛋白质构象变化来测量糖浓度，因此GGBP成为无试剂葡萄糖传感器开发的重要模型。 用差示扫描量热法(DSC)、远紫外圆二色谱和本征Trp荧光研究了D-葡萄糖结合对GGBP蛋白在pH 7.0条件下热去折叠的影响。所有这三种技术都显示了可逆的热转变，10 mM D-葡萄糖产生的中点温度(TM)从50摄氏度上升到63摄氏度。在无葡萄糖和有葡萄糖存在的情况下，GGBP在DSC中都观察到一个不对称的吸热曲线，尽管每个吸热曲线由两个相隔约4℃的Tm值转变组成。在没有葡萄糖的情况下，蛋白质去折叠最好的描述是两个非理想的转变，这表明存在去折叠中间产物。在D-葡萄糖存在下，蛋白质的展开比没有配体时更具协同性，实验数据最适合于假设两个理想(两态)顺序转变的模型。因此，D-葡萄糖结合改变了GGBP蛋白质折叠/去折叠的性质，将这两个结构域连接起来，使蛋白质去折叠成为一个合作的、两个两态的过程。根据DSC结果估算了63℃时葡萄糖结合的结合常数为5.9E61/M。根据热诱导的Trp荧光变化，DSC测量中稳定性较低的结构域被确定为GGBP的C-末端结构域。

Substrate Peptide Interactions with the ClpA Hexamer. (GP, AG, MRM, SKS, JAR) ClpA is an ATP-dependent chaperone that forms a hexameric ring that alone has an unfoldase activity in vitro. Two of these rings flank the ClpP tetradecamer-forming ClpAP protease. In this complex, ClpA assists the proteolytic core by binding, unfolding, and translocating substrates to the proteolytic chamber. Thermodynamic parameters of ClpA-peptide interactions have been determined by isothermal titration calorimetry (ITC) using a known 11-amino acid substrate ANDENYALAA (SsrA) and alpha-SsrA and by fluorescence titrations using dansylated SsrA peptide (DNS-SsrA). The DNS-SsrA has been found to be competitively displaced by other substrates of ClpA. Peptides containing the sequences of different parts of known substrates of ClpA and ClpX were used for competition with DNS-SsrA in fluorescent studies. This technique allows identification of specific motifs responsible for recognition by ClpA. Fluorescence methods have also been used to study the effect of adaptor proteins, such as ClpS, on the ClpA-peptide interactions. The fact that peptide sequences from different substrates compete with DNS-SsrA suggests that they occupy or overlap the same binding site of the ClpA hexameric ring. Conformational Stability and Domain Coupling in Glucose/Galactose-Binding Protein. (GP, AG, SA, MS, MR) The monomeric D-Glucose/D-Galactose-Binding Protein (GGBP) from Escherichia coli is a periplasmic protein that serves as a high-affinity receptor for the active transport and chemotaxis towards both sugars. Tight binding of sugars by the binding site located in the cleft between the two domains of the GGBP is achieved by the formation of hydrogen bonds with the ligand. Glucose and galactose binding induces a hinge motion between the two domains of the GGBP protein. GGBP become an important model for reagentless glucose sensor development due to the opportunity to measure sugar concentration by monitoring induced protein conformational change. The effect of D-glucose binding on the thermal unfolding of the GGBP protein at pH 7.0 has been measured by differential scanning calorimetry (DSC), far-UV circular dichroism, and intrinsic Trp fluorescence. All three techniques reveal reversible, thermal transitions and a midpoint temperature (Tm) increase from 50 to 63 C produced by 10 mM D-glucose. Both in the absence and presence of glucose, a single asymmetric endotherm for GGBP is observed in DSC, although each endotherm consists of two transitions about 4 C apart in Tm values. In the absence of glucose, the protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. In the presence of D-glucose, protein unfolding is more cooperative than in the absence of the ligand, and the experimental data are best fitted to a model that assumes two ideal (two-state) sequential transitions. Thus, D-glucose binding changes the character of the GGBP protein folding/unfolding by linking the two domains such that protein unfolding becomes a cooperative, two two-state process. An association constant of 5.9 E6 1/M at 63 C for glucose binding is estimated from DSC results. The domain with the lower stability in DSC measurements has been identified as the C-terminal domain of GGBP from thermally induced Trp fluorescence changes.