ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
批准号:
6085175
负责人:
BARBARA A SEATON
金额:
$11.53万
依托单位国家:
美国
项目类别:
财政年份:
1990
资助国家:
美国
项目状态:
已结题
起止时间:
1990-07-01 至 1999-12-02
关键词:
X ray crystallography amphiphilicity annexins calcium calcium channel chemical binding conformation crosslink crystallization fluorescence spectrometry hydropathy lectin liposomes membrane activity membrane proteins membrane reconstitution /synthesis nuclear magnetic resonance spectroscopy phospholipids phosphorylation physical model protein purification protein structure function site directed mutagenesis solutions structural biology
中文摘要
在细胞膜-细胞质界面上活跃的蛋白质在
独特的两相环境及其分子机制代表着一种
当前细胞生物学的前沿。膜联蛋白是一个界面蛋白家族
钙依赖的膜结合蛋白广泛分布于
真核生物。发现大量的(1-2%的细胞总蛋白)在许多
在组织中,膜联蛋白以水溶和膜结合的形式稳定存在。
对于结构生物学家来说,膜联蛋白提供了一个不同寻常的研究机会
无数的蛋白质介导的膜过程。Annexin V,一个推定的
钙跨膜移动的效应器,提供了一种极好的
用于研究膜联蛋白家族和其他界面的结构原型
蛋白质。这项研究计划的长期目标是开发一种
膜上膜联蛋白行为的综合图像。首要目标
建议的研究内容之一是开发膜结合的分子模型
Annexin V,详细说明其与膜组件的相互作用,以及
表征其对膜参数的影响。X射线结晶学和
解决方法将被用作研究两者的互补方法
膜联蛋白V的水溶性和膜结合型。
这一建议是为了:1)用结晶学方法研究膜联蛋白配体
相互作用;1a)与钙、脂类、
和磷脂类似物结合;1b)确定
具有一系列脱酰化或短链的膜联蛋白V络合物
磷脂酰丝氨酸化合物;1c)解决非磷脂结构
与Annexin V分子间疏水位点结合的化合物
晶体;1D)确定碳水化合物-膜联蛋白V复合体的结构。2)
通过突变研究膜联蛋白V的性质,使用结晶学和
溶液分析(蛋白质-囊泡结合、化学交联、核磁共振
光谱学,Fura-2-荧光测定法):2A)A K在非
有功能的钙结合环,看看钙结合能否恢复;
2B)E产生V突变,导致钙结合丢失
功能环,以查看结果对磷脂头部基团的影响
在这个位点结合;2c)假定决定因素的替代,见
膜联蛋白V的脂质结合偏好能否改变为
膜联蛋白I,探讨磷脂专一性的结构基础;2D)
破坏三聚体形成的突变,来研究它们之间的关系
齐聚和其他膜联蛋白特性。3)结晶,以便进一步
研究,另外两个膜联蛋白;3a)磷酸化的膜联蛋白IV,以研究
此修改的结构后果;以及3b)Annexin XI,
与Annexin V不同的是,它具有广泛的N-末端结构域。信息来自
这些研究将大大增加相当稀少的关于
界面蛋白,为膜联蛋白提供统一的结构基础
在膜上的作用,并提出了推测的结构基础
膜联蛋白V的功能可能与肾、肺、肝等器官有关
健康和疾病中的组织。
英文摘要
Proteins that are active at cell membrane-cytosol interfaces operate in a
unique, two-phase environment, and their molecular mechanisms represent a
current frontier of cell biology. Annexins are a family of interfacial
calcium-dependent membrane-binding proteins that are widely distributed in
eukaryotes. Found in very large amounts (1-2% total cell protein) in many
tissues, annexins exist stably in water-soluble and membrane-bound forms.
For structural biologists, annexins present an unusual opportunity to study
a myriad of protein-mediated membrane processes. Annexin V, a putative
effector of calcium movement across membranes, has provided an excellent
structural prototype for study of the annexin family and other interfacial
proteins. The long-term goal of this research program is to develop an
integrated picture of annexin behavior at the membrane. The primary goal
of the proposed research is to develop a molecular model of membrane-bound
annexin V, detail its interactions with membrane components, and
characterize its effects on membrane parameters. X-ray crystallography and
solution methods will be used as complementary approaches to study both
water-soluble and membrane-bound forms of annexin V. The specific aims of
this proposal are to; 1) Crystallographically study annexin-ligand
interactions; 1 a) Refine solved crystal structures with calcium, lipid,
and phospholipid analogues bound; 1b) determine crystal structures of
annexin V complexes with a series of deacylated or short-chain
phosphatidylserine compounds; 1 c) solve structures of non-phospholipid
compounds bound to an intermolecular hydrophobic site in annexin V
crystals; 1d) determine structures of carbohydrate-annexin V complexes. 2)
Investigate annexin V properties by mutagenesis, using crystallography and
solution assays (protein-vesicle binding, chemical cross-linking, NMR
spectroscopy, fura-2-fluorimetry): 2a) A K yields E mutation in a non-
functional calcium-binding loop to see if calcium-binding can be restored;
2b) an E yields V mutation causing loss of calcium binding in an otherwise
functional loop, to see the resulting effect on phospholipid head-group
binding at this site; 2c) substitution of putative determinants to see
whether the lipid binding preference of annexin V can be changes to that of
annexin I, to probe the structural basis of phospholipid specificity; 2d)
mutations that disrupt trimer formation, to study the relationship between
oligomerization and other annexin properties. 3) Crystallize, for further
study, two other annexins; 3a) phosphorylated annexin iV, to study the
structural consequences of this modification; and 3b) annexin XI, which,
unlike annexin V, has an extensive N-terminal domain. Information from
these studies will add much to the rather sparse body of knowledge about
interfacial proteins, provide a unifying structural basis for annexin
action at he membrane, and propose the structural basis for putative
annexin V function as it may relate to kidney, lung, liver, and other organ
tissues in health and disease.
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批准号:7695978
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资助金额:$203.58万
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财政年份:2009
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财政年份:2000
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财政年份:1999
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CA BRIDGING MECHANISM & PHOSPHOLIPID HEAD GROUP RECOGNITION IN ANNEXIN V
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资助金额:$0.0万
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财政年份:1998
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:2182572
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项目类别:
-
资助金额:$22.45万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:6476516
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项目类别:
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资助金额:$26.17万
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION
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批准号:3468143
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项目类别:
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资助金额:$10.31万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:2444753
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项目类别:
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资助金额:$23.13万
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:2904412
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项目类别:
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资助金额:$27.2万
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财政年份:1990
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负责人:BARBARA A SEATON
-
依托单位:
ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION
-
批准号:3468145
-
项目类别:
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资助金额:$11.9万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:6625079
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项目类别:
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资助金额:$26.94万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION
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批准号:2182570
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项目类别:
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资助金额:$12.76万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:2734659
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项目类别:
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资助金额:$24.04万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION
-
批准号:3468144
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项目类别:
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资助金额:$11.23万
-
财政年份:1990
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负责人:BARBARA A SEATON
-
依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:6329723
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项目类别:
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资助金额:$25.42万
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财政年份:1990
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负责人:BARBARA A SEATON
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依托单位:
ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS
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批准号:2182571
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项目类别:
-
资助金额:$23.82万
-
财政年份:1990
-
负责人:BARBARA A SEATON
-
依托单位:
ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION
-
批准号:3468146
-
项目类别:
-
资助金额:$11.82万
-
财政年份:1990
-
负责人:BARBARA A SEATON
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依托单位:
海外基金