利用核磁共振光谱法探究枯草芽孢杆菌复制终止蛋白与DNA的相互作用。

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy.

作者信息

Hastings Adam F, Otting Gottfried, Folmer Rutger H A, Duggin Iain G, Wake R Gerry, Wilce Matthew C J, Wilce Jacqueline A

机构信息

School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.

出版信息

Biochem Biophys Res Commun. 2005 Sep 23;335(2):361-6. doi: 10.1016/j.bbrc.2005.07.082.

DOI:10.1016/j.bbrc.2005.07.082

PMID:16061201

Abstract

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, alpha3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

摘要

枯草芽孢杆菌中DNA复制的终止涉及到二聚体29 kDa复制终止蛋白（RTP）与DNA终止位点之间形成的特定复合物对复制叉的极性阻滞。我们利用核磁共振光谱来探测添加21个碱基对对称DNA结合位点后，15N标记的RTP.C110S突变体的1H-15N相关光谱的变化。使用一套三重共振核磁共振实验对1H-15N相关性进行了归属，该实验使用了在800 MHz下记录的15N、13C、70% 2H富集的蛋白质，并使用了TROSY脉冲序列。对1H-15N光谱的扰动表明，N端、α3螺旋和几个环受到结合相互作用的影响。根据晶体学确定的RTP.C110S的无配体和DNA结合形式对该数据进行分析，结果表明，核磁共振光谱扰动与复合物形成时的蛋白质结构变化更密切相关，而不是与蛋白质-DNA界面的相互作用相关。