FLUORESCENT MOLECULAR ROTORS - A NEW CLASS OF PROBES FOR TUBULIN STRUCTURE AND ASSEMBLY
FLUORESCENT MOLECULAR ROTORS - A NEW CLASS OF PROBES FOR TUBULIN STRUCTURE AND ASSEMBLY
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DOI：
10.1021/bi00442a022
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发表时间：
1989-08-08
期刊：
影响因子：
2.9
通讯作者：
REED, JK
中科院分区：
文献类型：
--
作者：
KUNG, CE;REED, JK
9-（双氰乙烯基）julolidine （DCVJ）是一种分子内旋转弛豫依赖于溶剂的荧光染料。由于其量子产率随自由体积的减小而增加，该分子在监测合成聚合物反应和测量磷脂双层中局部微粘度变化方面非常有用[Loutfy， R. O. (1986) Pure appll。化学，58,1239-1248；孔志强，李志强（1986）生物化学[j]。我们已经使用DCVJ来跟踪微管蛋白的聚合，微管蛋白是一种可以组装成各种多态微结构的蛋白质。DCVJ与游离微管蛋白的结合伴随着量子产率的增加，表明DCVJ已经部分固定化。为4.度。C， DCVJ与单个高亲和疏水位点结合（Kd = 1.12 +-）。0.26 .mu。M)与蛋白质浓度相关的化学计量学。n，表示每摩尔。的DCVJ键的摩尔数。二聚体，浓度接近1。0.5 mg/mL，但降低到下限。apprxeq。0.3在浓度。gtoreq.2.0 mg/mL。量子产率也随蛋白质浓度的增加而增加。这种趋势不会因微管相关蛋白的存在而改变。这些结果是根据微管蛋白在4度的浓度依赖性寡聚化来分析的。C.微管蛋白在37度聚合时。在Zn2+存在的情况下，DCVJ的荧光强度增加，但增加的幅度有显著差异。我们能够利用结合染料的独特荧光和结合特性在分子水平上区分这两种多态。
9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., and Reed, J. K (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4.degree. C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 .+-. 0.26 .mu.M) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of .alpha..beta. dimer, approaches 1 at concentrations .ltoreq. 0.5 mg/mL but decreases to a lower limit of .apprxeq. 0.3 at concentrations .gtoreq.2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4.degree. C. When tubulin is polymerized at 37.degree. C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.