Analytical ultracentrifugation studies of oligomerization and DNA-binding of TtCarH, a Thermus thermophilus coenzyme B12-based photosensory regulator
Analytical ultracentrifugation studies of oligomerization and DNA-binding of TtCarH, a Thermus thermophilus coenzyme B12-based photosensory regulator
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DOI：
10.1007/s00249-013-0897-x
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发表时间：
2013-06-01
影响因子：
2
通讯作者：
Garcia de la Torre, Jose
中科院分区：
文献类型：
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作者：
Diez, Ana I.;Manuel Ortiz-Guerrero, Juan;Garcia de la Torre, Jose
Thermus thermophilus转录因子TtCarH是一类新发现的以5‘-脱氧腺苷钴胺(ADOb(12))为感光发色团的感光体。光调节依赖于ADOb(12)结合的低聚物在黑暗中的阻遏活性，光通过ADOb(12)光解导致的低聚物破坏来抵消光。在这项研究中，我们使用分析超速离心法(AUC)研究了TtCarH在黑暗和光照下与DNA的自结合和结合，包括沉降速度(SV)和平衡(SE)。从方法论的角度来看，这项研究表明，在光线是溶液性质的关键决定因素的情况下，AUC可以提供流体动力学方面的见解。对于光敏的TtCarH，吸光度以及干涉AUC数据也产生了类似的结果。SV实验的沉降系数和全身流体动力学分析表明，在溶液中，apo-TtCarH和光照下的ADOb(12)-TtCarH主要是非球形、椭圆形的单体，与SE数据一致。相比之下，ADOb(12)-TtCarH在黑暗中以更紧密的四聚体形式存在，较小的形式如二聚体或单体仍未被检测到，在较高的蛋白质浓度下出现少量较大的低聚物。AUC分析表明，在黑暗中，ADOb(12)-TtCarH以四聚体的形式与DNA结合，但在载脂蛋白形式或暴露于光下形成较小的络合物。由AUC推算出的TtCarH的自缔合和DNA结合特性与大小排斥和DNA结合凝胶位移分析的结果一致。AUC分析与流体动力学模拟相结合，提供了对TtCarH的ADOb(12)和光依赖的自结合和DNA结合的见解。
Thermus thermophilus transcriptional factor TtCarH belongs to a newly discovered class of photoreceptors that use 5'-deoxyadenosylcobalamin (AdoB(12)) as the light-sensing chromophore. Photoregulation relies on the repressor activity of AdoB(12)-bound oligomers in the dark, which light counteracts by oligomer disruption due to AdoB(12) photolysis. In this study, we investigated TtCarH self-association and binding to DNA in the dark and in the light using analytical ultracentrifugation (AUC) methods, both sedimentation velocity (SV) as well as equilibrium (SE). From a methodological point of view, this study shows that AUC can provide hydrodynamic insights in cases where light is a crucial determinant of solution properties. For the light-sensitive TtCarH, absorbance as well as interference AUC data yielded comparable results. Sedimentation coefficients and whole-body hydrodynamic analysis from SV experiments indicate that in solution apo-TtCarH and light-exposed AdoB(12)-TtCarH are predominantly aspherical, ellipsoidal monomers, in accord with SE data. By comparison, AdoB(12)-TtCarH exists as a more compact tetramer in the dark, with smaller forms such as dimers or monomers remaining undetected and low levels of larger oligomers appearing at higher protein concentrations. AUC analyses indicate that in the dark AdoB(12)-TtCarH associates as a tetramer with DNA but forms smaller complexes in the apo form or if exposed to light. The self-association and DNA-binding properties of TtCarH deduced from AUC are consistent with data from size-exclusion and DNA-binding gel-shift assays. AUC analyses together with hydrodynamic modeling provide insights into the AdoB(12)- and light-dependent self-association and DNA-binding of TtCarH.