Miki M
Department of Anatomy, University of Sydney, New South Wales, Australia.
Biochemistry. 1991 Nov 12;30(45):10878-84. doi: 10.1021/bi00109a011.
The distance between 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride or DNS-Cl) attached to Tyr-69 and N-[[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) or N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys-374 in an actin monomer was measured to be 2.51 nm or 2.27 +/- 0.04 nm, respectively, by fluorescence resonance energy transfer. This distance does not change significantly when the actin monomer binds DNase I, when the monomer is polymerized, when the polymer interacts with myosin subfragment 1, or when it interacts with tropomyosin-troponin in the presence and absence of Ca2+. Changes in the distance were within 0.1 nm. The results indicate that the structure of the region involving Tyr-69 and Cys-374 is substantially rigid. A large blue shift (about 15 nm) of the fluorescence spectrum and a large increase (about 80%) in the fluorescence intensity of DNS-actin were observed when DNS-actin was denatured upon addition of EDTA. On the other hand, a red shift (about 7 nm) of the fluorescence spectrum and a large decrease (about 50%) in the fluorescence intensity were observed when DNS-actin was completely unfolded in 8 M urea. The results indicate that dansyl chromophore becomes less exposed to the aqueous environment by EDTA denaturation in contradiction to the case of intrinsic tryptophan residues in G-actin. Resonance energy transfer measurements showed that the distance between probes attached to Tyr-69 and Cys-374 on an actin monomer changes by 0.37 nm during EDTA denaturation, but that the distance becomes longer than 4.0 nm in 8 M urea in which no energy transfer is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
通过荧光共振能量转移测量发现，肌动蛋白单体中与酪氨酸 - 69相连的5 - （二甲氨基）萘 - 1 - 磺酰氯（丹磺酰氯或DNS - Cl）和与半胱氨酸 - 374相连的N - [[4 - [4 - （二甲氨基）苯基]偶氮]苯基]马来酰亚胺（DABMI）或N - [4 - （二甲氨基） - 3,5 - 二硝基苯基]马来酰亚胺（DDPM）之间的距离分别为2.51纳米或2.27±0.04纳米。当肌动蛋白单体结合DNase I、单体聚合、聚合物与肌球蛋白亚片段1相互作用或在有或无Ca2 +存在的情况下与原肌球蛋白 - 肌钙蛋白相互作用时，该距离没有显著变化。距离变化在0.1纳米以内。结果表明，涉及酪氨酸 - 69和半胱氨酸 - 374的区域结构基本刚性。当加入EDTA使DNS - 肌动蛋白变性时，观察到荧光光谱有大的蓝移（约15纳米）且DNS - 肌动蛋白的荧光强度大幅增加（约80%）。另一方面，当DNS - 肌动蛋白在8 M尿素中完全展开时，观察到荧光光谱有红移（约7纳米）且荧光强度大幅降低（约50%）。结果表明，与G - 肌动蛋白中内在色氨酸残基的情况相反，丹磺酰发色团通过EDTA变性变得较少暴露于水环境。共振能量转移测量表明，在EDTA变性过程中，肌动蛋白单体上与酪氨酸 - 69和半胱氨酸 - 374相连的探针之间的距离变化0.37纳米，但在8 M尿素中该距离变得长于4.0纳米，此时未观察到能量转移。（摘要截短于250字）
