Sharma Alok K, Sawhney Paramvir, Memisoglu Gonen, Rigby Alan C
Center for Vascular Biology Research, Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215, United States.
Center for Vascular Biology Research, Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215, United States.
Protein Expr Purif. 2012 Jan;81(1):126-135. doi: 10.1016/j.pep.2011.09.013. Epub 2011 Oct 7.
Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.
MBS与PKG之间的蛋白质-蛋白质相互作用是由MBS的C末端结构域（MBS(CT180)）以及PKG-Iα的N末端卷曲螺旋（CC）亮氨酸拉链（LZ）结构域（PKG-Iα1(-59)）介导的。MBS(CT180)由三个结构不同的结构域组成，分别是非CC、CC和LZ性质的结构域。这些MBS结构域缺乏三维结构信息，妨碍了对MBS-PKG收缩复合物结构的原子水平理解。在此，我们展示了关于MBS(CT180)的CC、LZ和CCLZ结构域的克隆、表达和纯化的数据，以及使用尺寸排阻色谱（SEC）、圆二色性（CD）和二维（1）H-（15）N HSQC NMR对其进行的生物物理表征。详细描述的方法实现了高水平的蛋白质表达，并获得了毫克级数量的纯化的同位素（（15）N和（13）C）富集多肽。SEC、CD和（1）H-（15）N HSQC NMR实验表明，重组表达的MBS CC结构域折叠良好，在生理pH范围内以二聚体形式存在，这得到了我们先前研究结果的支持。CC MBS的二聚化可能是通过形成卷曲螺旋构象介导的。相比之下，MBS LZ结构域几乎未折叠，在生理pH范围内以不稳定的低结构单体形式存在。随着pH值降低，MBS LZ的蛋白质折叠和稳定性得到改善，在酸化至pH 4.5时采用折叠、稳定且有结构的构象。对LZ与CCLZ MBS结构域的SEC和NMR分析表明，包含CC结构域部分改善了LZ结构域的蛋白质折叠。
