Marabotti Anna, Herman Petr, Staiano Maria, Varriale Antonio, de Champdoré Marcella, Rossi Mosè, Gryczynski Zygmunt, D'Auria Sabato
Laboratory of Bioinformatics, Institute of Food Science, CNR, Avellino, Italy.
Proteins. 2006 Jan 1;62(1):193-201. doi: 10.1002/prot.20753.
The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.
通过稳态和时间分辨荧光光谱研究了压力对大肠杆菌D-半乳糖/D-葡萄糖结合蛋白（GGBP）结构和稳定性的影响，并研究了葡萄糖配体稳定GGBP结构的能力。稳态荧光实验表明，在没有葡萄糖的情况下，GGBP的荧光发射显著猝灭。相反，在低至中等压力值下，葡萄糖的存在似乎能稳定GGBP的结构。时间分辨荧光测量表明，在没有葡萄糖的情况下，GGBP的平均弛豫时间在高达600巴时变化显著，而在有配体存在时，在高达600巴的压力增加下它几乎不受影响。还通过分子动力学模拟研究了压力对GGBP的影响。模拟数据支持光谱结果，并证实葡萄糖的存在能够对抗压力对蛋白质结构的负面影响。综合来看，光谱和计算机模拟研究表明，在高达2000巴的压力值下，没有葡萄糖时GGBP的结构仍保持折叠状态，但可以检测到蛋白质二级结构的显著扰动。葡萄糖的结合降低了压力对蛋白质结构的负面影响，并赋予其免受扰动的保护，尤其是在中等压力值下。
