多肽诱导的大肠杆菌DegP(HtrA)蛋白酶的构象变化

具有分子伴侣和蛋白酶双重活性的大肠杆菌DegP蛋白,在热休克和其他应激条件下,对于降解和清除膜间质中变性或损伤的蛋白质起着十分重要的作用.到目前为止,已有几种蛋白质被鉴定出是DegP的天然底物.以前的研究表明,DegP的体内底物之一,PapG菌毛蛋白的羧基端多肽能够激活DegP的蛋白酶活性.然而这种激活的机制及生理意义均未见报道.用合成的PapG菌毛蛋白的羧基端多肽对这种激活的机制进行了初步研究.结果表明,DegP与多肽结合后发生了可检测的构象变化.圆二色性光谱结果显示,结合多肽后DegP的二级结构和三级结构均发生了一定的变化.凝胶排阻层析和动态光散射实验也揭示出DegP分子在一定程度上变小.进一步实验表明,DegP在多肽存在下,其疏水表面和催化位点均有所暴露.荧光各向异性结果显示出DegP在结合多肽后其构象柔性降低.对上述结果的意义进行了探讨.

Peptide Induced Conformational Changes of E. coli DegP(HtrA) Protease

The DegP protein, functioning as both chaperone and protease, plays a critical role in degrading and removing denatured ordamaged proteins in the cellular envelope during heat shock and other stresses. So far, several proteins have been identified as itsnatural targets. A carboxyle-terminal peptide derived from the PapG pilus, one of the in vivo substrates for DegP, has been shown toactivate the protease. Nevertheless, neither the details nor the physiological implications of such activation have been studied. Theevidence that DegP undergoes conformational changes upon binding the peptide derived from C-terminal sequence of pilus subunitPapG has been presented. It demonstrated that upon binding this peptide, detectable changes can be observed for both secondary andtertiary structures of DegP, as examined by CD spectroscopy. Gel filtration and dynamic light scattering analysis also revealed that thesize of DegP becomes smaller to a minor extent. Moreover, both the hydrophobic surfaces and catalytic sites of DegP were found toexpose slightly in the presence of the peptide. Upon peptide binding, a less flexible and more rigid conformation of DegP was obtainedas analyzed by fluorescence anisotropy. The physiological implications of these observations for DegP are discussed.