Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.
Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.
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使用荧光相关光谱和同源 FRET 分析破译 CaMKII 多聚化。
DOI：
10.1016/j.bpj.2017.02.005
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发表时间：
2017
影响因子：
3.4
通讯作者：
Vogel,StevenS
中科院分区：
文献类型：
--
作者：
Sarkar,Pabak;Davis,KaitlinA;Puhl3rd,HenryL;Veetil,JitheshV;Nguyen,TuanA;Vogel,StevenS
虽然激酶通常由一个或两个亚基组成，但钙钙调蛋白（CaM）依赖性蛋白激酶II （CaMKII）由8-14个亚基组成，它们围绕中心核心成对排列。目前尚不清楚CaMKII全酶是否作为独立亚基的组装，作为催化对，或作为一个单一的单位。解决这个问题的一个策略是通过基因工程改造单体和二聚体CaMKII，并评估它们的活性与野生型（WT）全酶的比较。本文采用荧光相关光谱和同源fret分析相结合的技术来表征金星标记的CaMKIIα的组装突变体，以鉴定二聚体CaMKII。然后用光谱学比较了二聚体突变体、wt -全酶和单体CaMKII寡聚域缺失突变体对照中全酶的结构和功能在CaM激活后的变化。CaM触发了WT和二聚体CaMKII的水动力体积增加，而没有改变亚基化学计量或金星标记的催化结构域之间的净同源fret。生化分析表明，二聚体突变体对外源底物的激酶活性和内源T286自磷酸化的功能也与WT全酶相似。我们得出结论，CaMKII全酶的基本功能单元是成对的催化结构域。
While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8–14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIαto identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.
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具有自发荧光蛋白质的荧光相关光谱。
DOI：
10.1007/b102212
发表时间：
2005
期刊：
Advances in biochemical engineering/biotechnology
影响因子：
--
作者：
T. Kohl;P. Schwille
通讯作者：
P. Schwille
影响因子：
4.8
作者：
Vogel, Steven S.;van der Meer, B. Wieb;Blank, Paul S.
通讯作者：
Blank, Paul S.
影响因子：
4.8
作者：
Strack, S;Choi, S;Colbran, RJ
通讯作者：
Colbran, RJ
影响因子：
3.4
作者：
Eggeling, C;Kask, P;Jäger, S
通讯作者：
Jäger, S
影响因子：
4.6
作者：
Vámosi G;Mücke N;Müller G;Krieger JW;Curth U;Langowski J;Tóth K
通讯作者：
Tóth K