Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
Department of Physical Biochemistry, University of Potsdam, 14476 Potsdam, Germany.
Biomolecules. 2019 Mar 2;9(3):84. doi: 10.3390/biom9030084.
Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil⁻helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze⁻thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of α-helical structure and the increased α-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil⁻helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of α-helical structure during freezing induced dehydration.
冷调节蛋白（COR）15A 是一种无序蛋白（IDP），对于抗冻性非常重要。在冷冻诱导的细胞脱水过程中，COR15A 从无序状态转变为主要α-螺旋结构。我们测试了增加 COR15A 的螺旋度是否也能增加其保护功能。鉴定并突变了保守的甘氨酸残基为丙氨酸。使用核磁共振（NMR）光谱法鉴定了野生型（WT）COR15A 和突变体的残基特异性螺旋度变化。圆二色性（CD）光谱法用于监测随着三氟乙醇（TFE）和乙二醇浓度增加时的螺旋-无规卷曲转变。通过荧光光谱法研究了 COR15A 突变体在冷冻-解冻循环过程中对模型膜稳定性的影响。这些实验的结果表明，突变体具有更高含量的α-螺旋结构，增加的α-螺旋度提高了冷冻过程中的膜稳定性。TFE 和乙二醇诱导的螺旋-无规卷曲转变的比较支持我们的结论，即在水相溶液中增加 COR15A 的瞬态螺旋度可以提高其在冷冻过程中稳定膜的能力。总之，我们的结果表明，保守的甘氨酸残基对于维持 COR15A 的无序结构很重要，但也与冷冻诱导脱水过程中α-螺旋结构的形成兼容。
