Quantitative analysis of pheromone-binding protein specificity.
Quantitative analysis of pheromone-binding protein specificity.
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DOI：
10.1111/j.1365-2583.2012.01167.x
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发表时间：
2013-02
影响因子：
2.6
通讯作者：
Renthal R
中科院分区：
文献类型：
--
作者：
Katti S;Lokhande N;González D;Cassill A;Renthal R
许多信息素的水溶性非常低，给定量结合测量带来了实验困难。提出了一种新方法，使用 β-环糊精作为增溶剂和转移剂，确定配体与信息素结合蛋白 (OBP) 结合的热力学有效解离常数。该方法适用于 LUSH，这是一种结合信息素 11-cis vaccenylacetate (cVA) 的果蝇 OBP。通过测量 N-苯基-1-萘胺 (NPN) 结合以及 LUSH 色氨酸 123 (W123) 和 NPN 之间的福斯特共振能量转移来评估大肠杆菌中表达的 LUSH 的重折叠。 cVA 的结合通过 W123 荧光的猝灭来测量，作为 cVA 浓度的函数。 β-环糊精和 LUSH 之间 cVA 转移的平衡常数由关联平衡模型确定。该常数乘以 β-环糊精-cVA 解离常数，得出 LUSH-cVA 解离常数：~100 nM。还发现其他配体可猝灭 W123 荧光。丝蛾信息素 bobykol 的 LUSH 配体解离常数确定为约 200 nM，油酸甲酯的解离常数为约 90 nM。结果表明，LUSH的配体结合腔可以容纳多种具有强结合相互作用的配体。这对 Laughlin 等人提出的信息素受体模型的影响。 （细胞133：1255-65，2008）进行了讨论。
Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed.
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DOI：
10.1046/j.1432-1327.2001.01927.x
发表时间：
2001-02-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Briand, L;Nespoulous, C;Pernollet, JC
通讯作者：
Pernollet, JC
DOI：
10.1073/pnas.0501447102
发表时间：
2005-04-12
影响因子：
11.1
作者：
Leal, WS;Chen, AM;Tsuruda, JM
通讯作者：
Tsuruda, JM
DOI：
10.1016/s0006-291x(02)00185-7
发表时间：
2002-04-26
影响因子：
3.1
作者：
Ban, LP;Zhang, L;Pelosi, P
通讯作者：
Pelosi, P
影响因子：
4.8
作者：
Campanacci, V;Krieger, J;Tegoni, M
通讯作者：
Tegoni, M
DOI：
10.1073/pnas.251532998
发表时间：
2001-12-04
影响因子：
11.1
作者：
Horst, R;Damberger, F;Wüthrich, K
通讯作者：
Wüthrich, K