When one plus one does not equal two: fluorescence anisotropy in aggregates and multiply labeled proteins.
When one plus one does not equal two: fluorescence anisotropy in aggregates and multiply labeled proteins.
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当一加一不等于二时：聚集体和多重标记蛋白质中的荧光各向异性。
DOI：
10.1016/j.bpj.2014.02.020
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发表时间：
2014
影响因子：
3.4
通讯作者：
Zolmajd-Haghighi Z
中科院分区：
文献类型：
--
作者：
Zolmajd-Haghighi Z
多染料体系的荧光各向异性和极化行为是众所周知的。随着能量转移距离内同类荧光团数量的增加，同型fret及其随之产生的能量迁移导致荧光各向异性降低。当一个簇内的所有亚基都被荧光团饱和时，这种行为就很好理解了。然而，内源性单体和标记单体的不完全标记、结合位点的不完全饱和或光漂白会产生随机混合物。广泛和长期使用的描述这些混合物的模型采用韦伯和丹尼尔于1966年首次提出的所有位点的荧光效率相等的假设。假设指出，当荧光团在溶液中自由时，它们在簇中彼此靠近时具有相同的亮度。该假设简化了对各向异性趋势的描述，因为簇的分数标记发生了变化。然而，近距离的荧光团往往由于自猝灭行为或外络合物形成等原因而表现出不可加性。因此，特定尺寸的荧光团簇的随机混合物的各向异性将取决于这些荧光团簇中的行为。我们提出了分数标记簇的分析表达式，展示了一系列的行为，以及两个系统的实验结果：一个组装的四聚体荧光蛋白簇和随机标记的牛血清白蛋白含有多达24个荧光团。实验结果表明，聚集的物种不遵循假设相同的荧光效率的系统研究与聚集的荧光团显示降低的荧光强度。应用等荧光效率的假设会低估各向异性，从而低估这两种情况下的簇大小。理论结果表明，在强淬火系统中仔细选择分数标记将增加确定簇大小的机会，使可访问的簇比目前认为可能的更大。
The behavior of fluorescence anisotropy and polarization in systems with multiple dyes is well known. Homo-FRET and its consequent energy migration cause the fluorescence anisotropy to decrease as the number of like fluorophores within energy transfer distance increases. This behavior is well understood when all subunits within a cluster are saturated with fluorophores. However, incomplete labeling as might occur from a mixture of endogenous and labeled monomer units, incomplete saturation of binding sites, or photobleaching produces stochastic mixtures. Models in widespread and longstanding use that describe these mixtures apply an assumption of equal fluorescence efficiency for all sites first stated by Weber and Daniel in 1966. The assumption states that fluorophores have the same brightness when free in solution as they do in close proximity to each other in a cluster. The assumption simplifies descriptions of anisotropy trends as the fractional labeling of the cluster changes. However, fluorophores in close proximity often exhibit nonadditivity due to such things as self-quenching behavior or exciplex formation. Therefore, the anisotropy of stochastic mixtures of fluorophore clusters of a particular size will depend on the behavior of those fluorophores in clusters. We present analytical expressions for fractionally labeled clusters exhibiting a range of behaviors, and experimental results from two systems: an assembled tetrameric cluster of fluorescent proteins and stochastically labeled bovine serum albumin containing up to 24 fluorophores. The experimental results indicate that clustered species do not follow the assumption of equal fluorescence efficiency in the systems studied with clustered fluorophores showing reduced fluorescence intensity. Application of the assumption of equal fluorescence efficiency will underpredict anisotropy and consequently underestimate cluster size in these two cases. The theoretical results indicate that careful selection of the fractional labeling in strongly quenched systems will enhance opportunities to determine cluster sizes, making accessible larger clusters than are currently considered possible.
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Homo-FRET 成像作为量化蛋白质和脂质聚类的工具
影响因子：
3.4
作者：
H. Gerritsen;A. Bader;E. Hofman;J. Voortman;P. Bergen;En Henegouwen;G. Meer
通讯作者：
G. Meer
影响因子：
3.9
作者：
D. Lidke;Peter Nagy;B. Barisas;Rainer Heintzmann;Janine N. Post;K. Lidke;A. H. Clayton;D. Arndt-Jovin;T. Jovin
通讯作者：
D. Lidke;Peter Nagy;B. Barisas;Rainer Heintzmann;Janine N. Post;K. Lidke;A. H. Clayton;D. Arndt-Jovin;T. Jovin
影响因子：
4.5
作者：
Matveeva, Evgenia G.;Terpetschnig, Ewald A.;Stevens, Megan;Patsenker, Leonid;Kolosova, Olga S.;Gryczynski, Zygmunt;Gryczynski, Ignacy
通讯作者：
Gryczynski, Ignacy
影响因子：
3.7
作者：
Permyakov S;Suzina N;Valiakhmetov A
通讯作者：
Valiakhmetov A
DOI：
10.1002/cyto.990100104
发表时间：
1989-01-01
期刊：
CYTOMETRY
影响因子：
--
作者：
MUJUMDAR, RB;ERNST, LA;WAGGONER, AS
通讯作者：
WAGGONER, AS