解析嗜热栖热菌35家族碳水化合物结合模块（CtCBM35）对葡萄糖和半乳糖取代甘露聚糖的配体特异性及其钙诱导稳定性。
Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for gluco- and galacto- substituted mannans and its calcium induced stability.
作者信息
Ghosh Arabinda, Luís Ana Sofia, Brás Joana L A, Pathaw Neeta, Chrungoo Nikhil K, Fontes Carlos M G A, Goyal Arun
机构信息
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
出版信息
PLoS One. 2013 Dec 6;8(12):e80415. doi: 10.1371/journal.pone.0080415. eCollection 2013.
This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×10(4) M(-1)), carob galactomannan (Ka = 12.4×10(4) M(-1)) and negligible association (Ka = 12 µM(-1)) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca(2+) ion with konjac glucomannan (Ka = 41×10(4) M(-1)) and carob galactomannan (Ka = 30×10(4) M(-1)). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a) with konjac glucomannan and carob galactomannan, 14.3×10(4) M(-1) and 11.4×10(4) M(-1), respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) -25 kJ mole(-1) as compared to carob galactomannan (ΔG) -22 kJ mole(-1). On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca(2+) ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.
本研究调查了来自热纤梭菌的CBM35（CtCBM35）在多糖识别中的作用。将CtCBM35克隆到带有工程化His6标签的pET28a(+)载体中，并在大肠杆菌BL21(DE3)细胞中表达。通过固定化金属离子色谱（IMAC）纯化得到一种均一的15 kDa蛋白。使用各种可溶性配体通过亲和电泳对CtCBM35进行配体结合分析。CtCBM35显示出对甘露糖构型配体的特异性结合，与魔芋葡甘露聚糖（Ka = 14.3×10⁴ M⁻¹）、角豆半乳甘露聚糖（Ka = 12.4×10⁴ M⁻¹）有显著结合，而与不溶性甘露聚糖的结合可忽略不计（Ka = 12 μM⁻¹）。CtCBM35与多糖的结合是钙依赖性的，在1 mM Ca²⁺存在下，与魔芋葡甘露聚糖（Ka = 41×10⁴ M⁻¹）和角豆半乳甘露聚糖（Ka = 30×10⁴ M⁻¹）的结合增加了两倍。通过荧光分光光度法研究进一步探究了多糖结合情况。与角豆半乳甘露聚糖和魔芋葡甘露聚糖结合时，CtCBM35的构象发生了显著变化，有规律地向较低量子产率方向发生21 nm的峰位移。与魔芋葡甘露聚糖和角豆半乳甘露聚糖的缔合度（Ka）分别为14.3×10⁴ M⁻¹和11.4×10⁴ M⁻¹，证实了亲和电泳的结果。与角豆半乳甘露聚糖相比，CtCBM35与魔芋葡甘露聚糖的结合导致更高的结合自由能（ΔG）-25 kJ·mol⁻¹（角豆半乳甘露聚糖为ΔG -22 kJ·mol⁻¹）。通过动态光散射（DLS）研究分析，CtCBM35与魔芋葡甘露聚糖和角豆半乳甘露聚糖结合时，流体力学半径（RH）分别从无配体时的4.25 nm增加到8 nm和6 nm。存在10 mM Ca²⁺离子时，CtCBM35颗粒的取向更硬，RH增加到4.52 nm。由于这种更硬的取向，CtCBM35变得更耐热，其解链温度从最初的50°C移至70°C。
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