Sequence effects of new N-capping motif CPxP on structural stability of YhhP protein

YhhP蛋白在细胞分裂中起作用，具有一个共同的序列基序CPxP。本研究旨在探讨该基序在Yhhp蛋白功能中的生物学和结构作用。为此，我们构建了包括CPxP在内的LRCPEP序列所有位置的一系列点突变体。对所有突变蛋白进行了功能生物测定，并在222 nm处用CD检测了它们对热、盐酸胍和尿素的稳定性。(1)点突变蛋白C19S和P20A/P22A完全缺陷，E21K部分缺陷，其余突变均未导致明显缺陷。YhhP的结构不受C19S突变的影响，但受到P20A/P22A双突变的强烈影响。因此，CPxP基序在结构和功能上都具有重要意义。(2)根据核磁共振的三维结构，在P20A/P22A中，A1-螺旋被延长了2个残基，而在P20A中，它被缩短了1个残基。这些结果与其他结果一起表明，LRCPEP确实形成了一种新型的N-帽基序。(3)参与蛋白质表面电荷变化的Arg18和Glu21的突变对蛋白质的稳定性有很大影响，认为YhP中Arg18和Glu21之间的静电相互作用较弱，但由于尿素和盐酸胍的作用，单个残基上电荷的变化显著地破坏了蛋白质的熔点Tm和变性中点。结果表明，Glu21的负电荷与螺旋偶极子的相互作用是该蛋白稳定性的关键。

YhhP protein is implemented in cell division and possesses a common sequence motif, CPxP. In this research project is to exploit biological and structural roles of this motif in YhhP protein function. To this end, we constructed a series of point mutants at all positions of sequence LRCPEP including CPxP. Functional bioassay was performed for all mutant proteins and their stability against heat, guanidine hydrochloride and urea was monitored by CD at 222nm.Research results were as follows. (1)Point mutant proteins C19S and P20A/P22A were completely defected and E21K was partially defected, and the rests of mutations did not result in significant defects. The structure of YhhP was not affected by C 19S mutation but strongly affected by double mutation of P20A/P22A. Thus, it was concluded that the CPxP motif is important both structurally and functionally. (2)According to the three-dimensional structure assessed by NMR, a1-helix was elongated by 2 residues in P20A/P22A, but it was shorten by one residue in P20A. Those results together with others indicated that LRCPEP indeed forms a new type of N-capping motif. (3)Mutations at the positions of Arg18 and Glu21 involved in changes in surface charge strongly affected protein stability, concluding that electrostatic interaction between Arg18 and Glu21 in YhhP is weak but that change of charges at individual residues destabilize significantly the protein in terms of melting point Tm and denaturation midpoints due to urea and guanidinehydrochloride. It was concluded that interaction of negative charge of Glu21 with the helix dipole is essential for stability of this protein YhhP.