Herman Petr, Barvik Ivan, Staiano Maria, Vitale Annalisa, Vecer Jaroslav, Rossi Mose', D'Auria Sabato
Faculty of Mathematics and Physics, Institute of Physics, Charles University, Ke Karlovu 5, 121 16 Prague, Czech Republic.
Biochim Biophys Acta. 2007 May;1774(5):540-4. doi: 10.1016/j.bbapap.2007.03.005. Epub 2007 Mar 20.
We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.
我们研究了温度对嗜热古菌嗜热栖热菌（TMBP）重组d -海藻糖/d -麦芽糖结合蛋白结合特异性的影响。重要的是，我们发现TMBP可以结合d -葡萄糖（Glc）。通过荧光光谱法在25℃至85℃的温度范围内对Glc结合进行了表征。我们的结果表明，在25℃时，Glc与TMBP的结合很好地由双峰模型表示，第一和第二结合步骤的表观解离常数（K(d)）分别为20μM和大约3 - 8 mM。在60℃时，Glc与TMBP的结合由表观K(d)值约为40μM的简单双曲线模型表示。最后，在85℃时Glc不与TMBP结合。使用分子动力学（MD）模拟来阐明Glc结合的分子机制。我们的结果表明，经过适当的荧光标记后，TMBP可以用作高度耐热且不消耗的分析物生物传感器，用于监测不存在其他糖类的液体（例如人体血液）中的葡萄糖水平。
