Herman Petr, Vecer Jaroslav, Barvik Ivan, Scognamiglio Viviana, Staiano Maria, de Champdoré Marcella, Varriale Antonio, Rossi Mose', D'Auria Sabato
Faculty of Mathematics and Physics, Institute of Physics, Charles University, Prague, Czech Republic.
Proteins. 2005 Oct 1;61(1):184-95. doi: 10.1002/prot.20582.
We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.
我们已对来自大肠杆菌的钙缺失型D-半乳糖/D-葡萄糖结合蛋白（GGBP）的稳定性和构象动力学进行了表征。通过稳态和时间分辨荧光以及在20摄氏度至70摄氏度温度范围内的远紫外圆二色性研究了该蛋白的结构稳定性。我们发现，Ca(2+)离子的缺失导致该蛋白C端结构域显著不稳定。特别是，解链温度降低了约10摄氏度，同时解链协同性丧失。时间分辨荧光猝灭显示，当高度屏蔽的色氨酸残基在较高温度下可被丙烯酰胺接近时，蛋白发生了显著的松散。我们记录了葡萄糖的显著稳定作用，其大多逆转了钙的作用，也就是说，蛋白的热稳定性提高了约10摄氏度，且解链协同性得以恢复。此外，直至高温时，蛋白结构仍保持紧密，片段迁移率的幅度较低。我们利用分子动力学来确定导致温度稳定性变化的结构特征。Ca(2+)结合环的解体似乎是导致在无钙情况下稳定性丧失的原因。对钙缺失型GGBP的结构特性和温度稳定性的新见解有助于更好地理解该蛋白的功能，并为开发这类蛋白的新生物技术应用提供重要信息。
