Calorimetric and fluorescence studies of protein propert

ClpA是形成六聚环的ATP依赖性分子伴侣，其单独在体外具有解折叠酶活性。这些环中的两个位于ClpP十四聚体的侧翼，形成ClpAP蛋白酶。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通过等温滴定量热法（ITC）使用已知的11-氨基酸底物ANDENYALAA（SsrA）和3betaSsrA测定。丹磺酰化SsrA（DNS-SsrA）被用于荧光滴定，通过一种独立的方法获得结合常数和结合化学计量。已发现DNS-SsrA也被ClpA的较大底物如RepA或3betaSsrA竞争性置换，但不被非结合肽AANDENYALDD（DD-SsrA）竞争性置换。与DNS-SsrA的竞争研究已用于定位已知ClpA底物的特异性结合序列。 来自大肠杆菌的葡萄糖/半乳糖结合蛋白（GGBP）是一种周质蛋白，其充当对两种糖配体的主动转运和趋血性的高亲和力受体。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。 我们研究了葡萄糖结合对大肠杆菌GGBP蛋白热稳定性的影响。通过差示扫描量热法（DSC）、圆二色性（CD）和Trp内源荧光光谱分析，研究了大肠杆菌中Trp基因的表达。所有这三种技术都揭示了10 mM D-葡萄糖对这两个结构域蛋白质展开的强烈稳定作用。

ClpA is an ATP dependent chaperone that forms a hexameric ring that alone has an unfoldase activity in vitro. Two of these rings flank 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 3betaSsrA. Dansylated SsrA (DNS-SsrA) has been used in fluorescence titrations to obtain the association constant and binding stoichiometry by an independent method. The DNS-SsrA has been found also to be competitively displaced by larger substrates of ClpA such as RepA or 3betaSsrA but not by the nonbinding peptide AANDENYALDD (DD-SsrA). Competition studies with DNS-SsrA has been used to locate specific binding sequences of known ClpA substrates. The Glucose/Galactose Binding Protein (GGBP) from Escherichia coli is a periplasmic protein that serves as a high-affinity receptor for the active transport and hemotaxis towards both sugar ligands. 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. We have investigated the effect of glucose binding on the thermal stability of the GGBP protein from E. coli by differential scanning calorimetry (DSC), circular dichroism (CD) and intrinsic Trp fluorescence. All three techniques reveal strong stabilizing effects of 10 mM D-glucose on the unfolding of this two-domain protein.