Calorimetric and fluorescence studies of protein propert

底物肽与ClpA六聚体的相互作用。(GP、AG、MRM、SKS、ESTA） ClpA是ATP依赖性分子伴侣，其形成六聚体环，其单独在体外具有解折叠酶活性。这些环中的两个位于形成ClpP十四聚体的ClpAP蛋白酶的侧翼。在该复合物中，ClpA通过将底物结合、解折叠和移位至蛋白水解室来辅助蛋白水解核心。ClpA-肽相互作用的热力学参数已经通过等温滴定量热法（ITC）使用已知的11-氨基酸底物ANDENYALAA（SsrA）和α-SsrA以及通过荧光滴定使用丹磺酰化SsrA肽（DNS-SsrA）来确定。已发现DNS-SsrA被ClpA的其他底物竞争性地置换。含有ClpA和ClpX的已知底物的不同部分的序列的肽用于在荧光研究中与DNS-SsrA竞争。该技术允许鉴定负责ClpA识别的特定基序。荧光方法也被用来研究衔接蛋白，如ClpS，对ClpA-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ClpA六聚体环的相同结合位点。 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） 来自大肠杆菌的单体D-葡萄糖/D-半乳糖结合蛋白（GGBP）是一种周质蛋白，其充当对两种糖的主动转运和趋化性的高亲和力受体。位于GGBP两个结构域之间的裂缝中的结合位点与糖的紧密结合通过与配体形成氢键来实现。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可以通过监测诱导蛋白质构象变化来测量糖浓度，因此成为无试剂葡萄糖传感器开发的重要模型。 已通过差示扫描量热法（DSC），远紫外圆二色性，和固有的色氨酸荧光D-葡萄糖结合的GGBP蛋白在pH 7.0的热展开的效果进行了测量。所有这三种技术都揭示了可逆的热转变和由10 mM D-葡萄糖产生的中点温度（Tm）从50 ℃升高到63 ℃。在葡萄糖存在和不存在下，在DSC中观察到GGBP的单一不对称吸热，尽管每个吸热由两个Tm值相距约4 C的跃迁组成。在没有葡萄糖的情况下，蛋白质展开最好由两个非理想的转变，这表明存在展开中间体。在D-葡萄糖的存在下，蛋白质展开比在没有配体的情况下更合作，并且实验数据最适合于假设两个理想的（两态）顺序转换的模型。因此，D-葡萄糖结合通过连接两个结构域改变GGBP蛋白折叠/解折叠的特征，使得蛋白解折叠成为一个合作的两个两态过程。从DSC结果估计63 ℃下葡萄糖结合的缔合常数为5.9 E6 1/M。在DSC测量中具有较低稳定性的结构域已被确定为GGBP的C-末端结构域，从热诱导的Trp荧光变化。

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.