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细丝的分子基础。
阵型和周转。肌动蛋白必须形成细丝才能完成其
生物角色。它必须与ATP或ADP结合,并与二价体结合
阳离子以保持其固有的结构。最后,水解物的
核苷酸伴随着聚合和随后的释放
PI产生ADP-F肌动蛋白,它是一种比ATP或ADP-更不稳定的细丝-
PI F肌动蛋白。因此,肌动蛋白细丝循环可能取决于PI的速率
从灯丝上松开。尽管已知G-肌动蛋白的结构
对于原子分辨率来说,丝状结构还没有直接得到
下定决心。Holmes等人。在此基础上提出了F-肌动蛋白模型
与光纤衍射数据相耦合的单体结构
定向肌动蛋白凝胶。该模型预测循环的重定向
在亚域3和亚域4之间,包含疏水“插头”,以允许
它与一个疏水口袋相互作用,该口袋由亚域2和
4在相反链中的相邻单体。这样一座桥将会
大大增强了结构的线间稳定性。然而,
该模型的这一方面尚未得到实验验证。我们有
通过定点突变改变酵母肌动蛋白的塞子开始
来检验即插即用相互作用理论。要继续,我们将放置
建议构象活性插头位置的半胱氨酸,L269。
我们将安装一个荧光探头并研究插头的行为
在G和F状态下使用稳态荧光和荧光
共振能量传递。我们将在H73上创造突变体来测试
提出将这一残留物作为决定
PI释放率。与冷敏聚合缺陷相关的
这些突变体表明它们可能会削弱亚区2的成分
即插即用交互的口袋。我们将通过以下方式进行测试
研究稳定这些突变细丝以对抗
寒冷的天气。我们将结合H73和L269C突变来检查
单体中的环在以下条件下的行为
通常是聚合的。最后,我们将使用遗传学来确定
蛋白质,在体内,恢复我们聚合的聚合性-
有缺陷的突变体。这项工作应该提供关于
F-肌动蛋白组装和拆解的分子基础。
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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会议论文
Biochemical consequences of Deafness-causing actin mutations
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批准号:7850295
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项目类别:
-
资助金额:$24.95万
-
财政年份:2009
-
负责人:Peter A. Rubenstein
-
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:
-
资助金额:$31.56万
-
财政年份:2007
-
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: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:
-
资助金额:$31.88万
-
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
-
项目类别:
-
资助金额:$26.88万
-
财政年份:2007
-
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
-
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: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:
A Mutagenic Study of Yeast Actin Conformational Changes
-
批准号:7037479
-
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
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批准号:6615986
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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:
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:
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
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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
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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