Protein-protein forster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z
Protein-protein forster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z
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DOI：
10.1016/j.jmb.2007.05.075
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发表时间：
2007-08-24
影响因子：
5.6
通讯作者：
Gloss, Lisa M.
中科院分区：
文献类型：
--
作者：
Hoch, Duane A.;Stratton, Jessica J.;Gloss, Lisa M.
设计了一个蛋白质-蛋白质福斯特共振能量转移（FRET）系统，该系统采用多个位置的探针，专门监测H2A-H2B二聚体与核小体核心粒子（NCP）的解离。选择色氨酸供体和Cys-AEDANS受体是因为，与以前的NCP FRET荧光团相比，它们：(1)更小，疏水性更低，这应该最大限度地减少组蛋白和NCP结构的扰动；和(2)的R-O为20埃，远小于NCP的尺寸（类似于50埃宽，类似于100埃直径）。平衡蛋白展开滴定表明，供体和受体部分对H2A-H2B二聚体和（H3-H4）(2)四聚体的稳定性影响很小。用601 DNA定位元件重组含有不同FRET对的ncp。nacl诱导的NCP平衡解离表明，相对于较弱的定位序列，601序列使NCP稳定为二聚体解离。这一发现表明H2A-H2B二聚体在确定NCP稳定性的DNA序列依赖性方面发挥了重要作用。从可逆和明确的s型转变中确定的解离自由能显示出两个不同的相，反映了单个H2A-H2B二聚体的解离，证实了先前提出的协同性；这些数据允许对协作性进行定量描述。然后使用FRET系统研究组蛋白变体H2A的影响。Z论NCP稳定性；以前的研究报告了不稳定和稳定的影响。H2A。Z FRET NCP离解转变表明稳定性略有增加，但二聚体离解的协同性显著增加。因此，这种蛋白质-蛋白质FRET系统用于监测组蛋白变异对NCP动力学的影响已被证明，该系统似乎同样适合于二聚体结合和NCP解离的动力学过程的解剖。(c) 2007 Elsevier Ltd.版权所有。
A protein-protein Forster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, compared to previous NCP FRET fluorophores, they: (1) are smaller and less hydrophobic, which should minimize perturbations of histone and NCP structure; and (2) have an R-O of 20 angstrom, which is much less than the dimensions of the NCP (similar to 50 angstrom width and similar to 100 angstrom diameter). Equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)(2) tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 DNA positioning element. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation relative to weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of individual H2A-H2B dimers, confirming cooperativity as suggested previously; these data allow quantitative description of the cooperativity. The FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity of the dimer dissociations. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer association and dissociation from the NCP. (c) 2007 Elsevier Ltd. All rights reserved.