Khrapunov Sergei, Cheng Huiyong, Hegde Subray, Blanchard John, Brenowitz Michael
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2008 Dec 26;283(52):36290-9. doi: 10.1074/jbc.M804702200. Epub 2008 Oct 31.
The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.
五肽重复序列是最近发现的一种蛋白质折叠结构。结核分枝杆菌的MfpA是五肽重复序列蛋白（PRP）家族的首个成员，它通过与DNA促旋酶结合并抑制其活性，赋予对氟喹诺酮类抗生素的抗性。MfpA的大小、形状和表面电势与双链DNA相似。作为对PRP在调节细胞拓扑异构酶活性及赋予抗生素抗性方面作用进行全面生物物理分析的第一步，我们通过荧光光谱法、圆二色光谱法（CD）和分析超速离心法研究了MfpA的溶液结构和重折叠过程。描述了五肽重复序列折叠结构独特的CD光谱。该光谱揭示了一种天然结构，其在定义PRP折叠结构的右手四边形β-螺旋内的β-链和转角与典型二级结构类型不同。通过透析或稀释从尿素或胍中重折叠的MfpA形成了单体的稳定聚集体，其二级和三级结构并非天然结构。相比之下，使用一种新颖的“时间依赖性复性”方案重折叠的MfpA产生了具有天然二级、三级和四级结构的蛋白质。文中还讨论了“时间依赖性复性”对其他蛋白质和变性方法的通用性。
