MUTAGENIC STUDY OF YEAST ACTIN CONFORMATIONAL CHANGES
MUTAGENIC STUDY OF YEAST ACTIN CONFORMATIONAL CHANGES
批准号:
6519149
负责人:
Peter A. Rubenstein
金额:
$30.19万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1984
资助国家:
美国
项目状态:
已结题
起止时间:
1984-07-01 至 2003-03-31
关键词:
Saccharomyces cerevisiae actins adenosine triphosphate adenosinetriphosphatase conformation cysteine fluorescence resonance energy transfer fungal genetics fungal proteins gene mutation hydrolysis hydropathy molecular cloning polymerization protein biosynthesis protein structure function site directed mutagenesis temperature sensitive mutant
中文摘要
我们正试图确定肌动蛋白丝的分子基础
形成和周转。 肌动蛋白必须形成细丝来完成其
生物角色。 它必须与ATP或ADP沿着以二价结合,
阳离子以保持其天然结构。最后,水解的
伴随着聚合和随后的核苷酸的释放,
Pi产生ADP-F肌动蛋白,这是一种比ATP或ADP更不稳定的细丝。
β F肌动蛋白。 因此,肌动蛋白丝循环可能取决于Pi的速率,
从灯丝中释放出来。 虽然G-肌动蛋白的结构是已知的,
到原子分辨率,细丝结构还没有直接
测定 Holmes等人提出了一个基于F-actin的模型,
单体的结构与纤维衍射数据相结合,
定向肌动蛋白凝胶。 该模型预测了一个环的重新定向
在亚结构域3和4之间,含有疏水性“塞”,以允许
它与由亚结构域2组成的疏水口袋相互作用,
4个相邻单体在相对链中。 这样一座桥,
大大增强了结构的股间稳定性。 然而,在这方面,
该模型的这一方面尚未得到实验验证。 我们有
通过定点突变改变了酵母肌动蛋白的塞子,使其开始
来验证塞袋相互作用理论 接下来,我们将
一个半胱氨酸在一个建议的构象活性插头的位置,L269。
我们将附加一个荧光探针并研究插头的行为
在G和F状态下使用稳态荧光和荧光
共振能量转移 我们将在H73上制造突变体,
理论提出这个剩余作为一个重要因素,在确定
Pi释放率 冷敏聚合缺陷与
这些突变体表明,它们可能会削弱亚结构域2的组成部分,
插塞-口袋相互作用的口袋。 我们将测试这一点,
研究稳定这些突变细丝所需的因素,
寒冷 我们将联合收割机结合H73和L269 C突变,
在单体中的环的行为,
正常情况下, 最后,我们将使用遗传学来确定
蛋白质,在体内,恢复我们的聚合能力,
有缺陷的突变体 这项工作应该提供新的见解,
F-actin组装和拆卸的分子基础。
英文摘要
We are trying to ascertain the molecular basis underlying actin filament
formation and turnover. Actin must form a filament to fulfill its
biological roles. It must bind to ATP or ADP along with a divalent
cation to maintain its native structure. Finally, the hydrolysis of the
nucleotide concomitant with polymerization and subsequent release of the
Pi creates ADP-F actin which is a less stable filament than ATP- or ADP-
Pi F actin. Thus, actin filament cycling may depend on the rate of Pi
release from the filament. Although the structure of G-actin is known
to atomic resolution, the filament structure has not been directly
determined. Holmes et al. proposed a model of F-actin based on the
structure of the monomer coupled with fiber diffraction data from
oriented actin gels. The model predicts a reorientation of a loop
between subdomains 3 and 4, containing a hydrophobic "plug", to allow
it to interact with a hydrophobic pocket composed of subdomains 2 and
4 of adjacent monomers in the opposing strand. Such a bridge would
greatly enhance interstrand stabilization of the structure. However,
this aspect of the model has not been experimentally verified. We have
altered the plug of yeast actin by site-directed mutagenesis to begin
to test the plug-pocket interaction theory. To continue, we will place
a cysteine in a proposed conformationally active plug position, L269.
We will attach a fluorescent probe and study the behavior of the plug
in the G and F states using steady state fluorescence and fluorescence
resonance energy transfer. We will create mutants at H73 to test a
theory proposing this residue as an important factor in determining the
rate of Pi release. The cold-sensitive polymerization defect associated
with these mutants suggests they may weaken the subdomain 2 component
of the pocket of the plug-pocket interaction. We will test this by
examining the factors needed to stabilize these mutant filaments against
the cold. We will combine the H73 and L269C mutations to examine the
behavior of the loop in the monomer under conditions where it would
normally polymerize. Finally, we will use genetics to determine which
proteins, in vivo, restore polymerizability to our polymerization-
defective mutants. This work should provide new insight concerning the
molecular basis underlying F-actin assembly and disassembly.
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Biochemical consequences of Deafness-causing actin mutations
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批准号:7850295
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项目类别:
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资助金额:$24.95万
-
财政年份:2009
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负责人:Peter A. Rubenstein
-
依托单位:
Biochemical Consequences of Deafness-causing Actin Mutations
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批准号:7476109
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项目类别:
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资助金额:$10.0万
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财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
Biochemical consequences of Deafness-causing actin mutations
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批准号:7384548
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项目类别:
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资助金额:$31.88万
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财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
Biochemical consequences of Deafness-causing actin mutations
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批准号:8009461
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项目类别:
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资助金额:$30.55万
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财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
Biochemical consequences of Deafness-causing actin mutations
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批准号:7738925
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项目类别:
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资助金额:$31.56万
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财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
Biochemical consequences of Deafness-causing actin mutations
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批准号:7534363
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项目类别:
-
资助金额:$26.88万
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财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
Biochemical consequences of Deafness-causing actin mutations
-
批准号:8197273
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项目类别:
-
资助金额:$30.55万
-
财政年份:2007
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负责人:Peter A. Rubenstein
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依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
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批准号:7037479
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项目类别:
-
资助金额:$34.57万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:7786244
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项目类别:
-
资助金额:$36.19万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
FUNCTIONAL SIGNIFICANCE OF THE ACTIN N-TERMINAL REGION
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批准号:3283604
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项目类别:
-
资助金额:$16.51万
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财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
ACTIN NH2-TERMINAL PROCESSING AND ITS SIGNIFICANCE
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批准号:3283601
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项目类别:
-
资助金额:$14.0万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:6615986
-
项目类别:
-
资助金额:$35.37万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO
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批准号:2177095
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项目类别:
-
资助金额:$21.43万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO
-
批准号:2684778
-
项目类别:
-
资助金额:$23.2万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
FUNCTIONAL SIGNIFICANCE OF THE ACTIN N-TERMINAL REGION
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批准号:2177093
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项目类别:
-
资助金额:$18.66万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
MUTAGENIC STUDY OF YEAST ACTIN CONFORMATIONAL CHANGES
-
批准号:2839791
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项目类别:
-
资助金额:$30.35万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:6878005
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项目类别:
-
资助金额:$35.4万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:7265349
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项目类别:
-
资助金额:$36.41万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO
-
批准号:2177094
-
项目类别:
-
资助金额:$22.43万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:7391220
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项目类别:
-
资助金额:$36.56万
-
财政年份:1984
-
负责人:Peter A. Rubenstein
-
依托单位:
海外基金