National Magnetic Resonance Facility at Madison, University of Wisconsin, Madison, Wisconsin 53705, USA.
J Biol Chem. 2011 Dec 16;286(50):43447-53. doi: 10.1074/jbc.M111.306464. Epub 2011 Oct 13.
Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R(1) and R(2), relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.
Hsp12（热休克蛋白 12）属于小热休克蛋白家族，部分特征为应激反应、停滞期进入、晚期胚胎丰富样蛋白，位于质膜上，以防止膜干燥。在这里，我们通过 NMR 和生物物理技术报告了 Hsp12 的结构特征。该蛋白用氮-15 和碳-13 均匀标记，以便在水相和两种膜模拟环境（SDS 和十二烷基磷酸胆碱（DPC）胶束）中详细确定其构象。从分配的化学位移确定的二级结构元素表明，Hsp12 在水溶液中是动态无序的，而在 SDS 胶束存在下，它获得四个螺旋延伸，在 DPC 存在下获得一个单螺旋。这些结论通过该蛋白在所有三种环境中的圆二色性光谱得到了加强。NOESY 光谱中没有长程相互作用表明，SDS 胶束中存在的螺旋并未组装在一起。R1 和 R2、弛豫和异核 NOE 测量表明，该蛋白在水溶液中无序，但在存在去污剂胶束时变得更加有序。在顺磁自旋弛豫剂（5DSA、16DSA 和 Gd（DTPA-BMA））存在下收集的 NMR 光谱表明，Hsp12 在 SDS 胶束中的两亲性α-螺旋位于膜表面上。这些观察结果与表明 Hsp12 可防止膜干燥的研究结果一致。
