Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA:: Single molecule force spectroscopy measurements
Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA:: Single molecule force spectroscopy measurements
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DOI：
10.1016/j.jmb.2005.03.065
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发表时间：
2005-06-03
影响因子：
5.6
通讯作者：
Williams, MC
中科院分区：
文献类型：
--
作者：
Pant, K;Karpel, RL;Williams, MC
噬菌体T4基因32蛋白(Gp32)是单链DNA(SsDNA)结合蛋白大家族中研究较多的代表蛋白，在DNA复制、重组和修复中起重要作用。令人惊讶的是，以前还没有观察到gp32可以融化天然的dsDNA。同时，缺少C-末端结构域(CTD)的gp32的截短版本*I被证明能使天然DNA的熔化温度降低约50℃。鉴于先前测量到的这两种蛋白与单链DNA的结合是相似的，gp32和*I双链不稳定能力的这种深刻差异尤其令人费解。在这里，我们通过研究gp32和*i对双链DNA熔融的热力学和动力学的影响来解决这一明显的矛盾。我们使用以前发展的单分子技术来测量双链DNA的非合作结合常数(K-DS)，以确定KDS作为gp32和*I的盐浓度的函数。然后，我们发展了一种新的单分子方法来测量K，即这些蛋白质与单链DNA的结合常数。比较我们测得的gp32和*i与ssDNA的结合常数，我们发现虽然它们在高盐中非常相似，但在[Na+]<；0.2M时它们强烈发散。这些结果表明，完整蛋白质必须经历CTD的构象重排，该构象重排处于预平衡状态，与dsDNA和ssDNA的非合作结合。这降低了可用于结合的蛋白质的有效浓度，进而降低了它破坏dsDNA稳定的速度。第一次，我们量化了这种CTD展开的自由能，并表明它强烈地依赖于盐，并与CTD上的钠反离子凝聚有关。(C)2005年由爱思唯尔有限公司出版。
Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K-ds) to double-stranded DNA to determine Kds as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K, the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD. (c) 2005 Published by Elsevier Ltd.