Protein dynamics control proton transfer from bulk solvent to protein interior: A case study with a green fluorescent protein
Protein dynamics control proton transfer from bulk solvent to protein interior: A case study with a green fluorescent protein
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DOI：
10.1110/ps.051391205
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发表时间：
2005-07-01
期刊：
影响因子：
8
通讯作者：
Krishnamoorthy, G
中科院分区：
文献类型：
--
作者：
Saxena, AM;Udgaonkar, JB;Krishnamoorthy, G
绿色荧光蛋白（GFP）的质子转移动力学是表征蛋白质动力学与功能相关性的模型系统。通过使用激光诱导的pH跳跃方法监测EGFP（GFP的变体）中的动力学。通过纳秒闪光光解“笼状质子”邻硝基苯甲醛，将pH从8跳到5，并通过跟踪荧光强度的降低来监测随后的质子转移。质子转移动力学的调制外部扰动，如粘度，pH值，和亚变性浓度的GdnHCl以及盐进行了研究。质子转移的速率与溶剂粘度成反比，表明限速步骤是质子通过蛋白质基质的转移。速率在较低的pH值下加速，色氨酸57的荧光性质的测量表明，速率的增强与蛋白质动力学的增强有关。质子转移的速率几乎与温度无关，与反向过程的速率不同。当蛋白质的稳定性通过添加共溶质（包括盐KCl、KNO3和K2SO4）而降低或增加时，在所有情况下观察到质子转移速率的显著降低。质子转移速率和蛋白质稳定性之间缺乏相关性表明，结构被调整以确保控制蛋白质质子转移功能的动力学的最大效率。
The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO3, and K2SO4, a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.