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

YhhP蛋白在细胞分裂中实现，并且具有共同的序列基序CPxP。在本研究项目中，将利用该基序在YhhP蛋白功能中的生物学和结构作用。为此，我们构建了一系列的点突变体在所有位置的序列LRCPEP，包括CPxP。对所有突变蛋白进行了功能性生物测定，并采用CD法在222 nm处检测了突变蛋白对热、盐酸胍和尿素的稳定性。（1）点突变蛋白C19 S和P20 A/P22 A完全缺失，E21 K部分缺失，其余突变蛋白无明显缺陷。YhhP的结构不受C19 S突变的影响，但受P20 A/P22 A双突变的强烈影响。因此，可以得出结论，CPxP基序在结构和功能上都很重要。（2）通过核磁共振（NMR）分析，P20 A/P22 A的α 1-螺旋延长了2个残基，而P20 A的α 1-螺旋缩短了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-加帽基序。（3）Arg 18和Glu 21位点的突变引起表面电荷的变化，对蛋白质的稳定性有很大的影响，说明YhhP中Arg 18和Glu 21之间的静电相互作用很弱，但单个位点的电荷变化使蛋白质的熔点Tm和变性中点（由尿素和盐酸胍引起）明显不稳定。可以得出结论，Glu 21的负电荷与螺旋偶极子的相互作用是必不可少的这种蛋白质YhhP的稳定性。

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.