MECHANISM OF PROTEIN INSERTION INTO LIPID BILAYERS

蛋白质插入脂质双层的机制

基本信息

批准号：
3294222
负责人：
MARTIN TEINTZE
金额：
$ 8.03万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 1994-03-31
项目状态：
已结题

项目摘要

The mechanisms by which proteins are integrated into or translocated across cell membranes are subjects of major interest in modern molecular and cell biology. Studies on a number of integral membrane proteins have shown that they can spontaneously incorporate into phospholipid bilayers without the presence of any other proteins to mediate this insertion. The object of the proposed research is to determine which features of the primary and secondary structure of an integral membrane protein allow it to insert into a phospholipid bilayer. This will be investigated using a model the spontaneous insertion of bacteriorhodopsin fragments and mutants thereof into performed unilamellar vesicles of dimyristoylphosphatidylcholine. Bacteriorhodopsin, which has the most well-studied structure of any membrane protein, consists seven helical domains crossing back and forth across the membrane bilayer connected by hydrophlic sequences of varying length. Preliminary studies have shown that when the protein is cleaved with chymotrypsin between domains 2 and 3, the larger fragment which results is still capable of membrane insertion. Additional fragments containing various combinations of domains and mutants thereof will now be generated both by proteolysis and by manipulation of the cloned gene in an expression vector using site-specific mutagensis. Incorporation experiments using these polypeptides and phospholipid vesicles will be used to narrow down which sequences in bacteriorhodoposin are necessary and sufficient for insertion into a phospholipid bilayer. Results from these experiments will form the basis for studies using additional mutations to determine what structures mediate membrane insertion and which determine the orientation in the membrane. Finally the sequences identified as mediating insertion of an integral membrane protein will be tested to determine whether they can also anchor or translocate an otherwise hydrophilic protein attached to them.

蛋白质整合或整合的机制跨细胞膜易位是主要关注的主题在现代分子和细胞生物学中。研究了多项整合膜蛋白已表明它们可以自发地融入磷脂双分子层中，无需任何其他蛋白质的存在来介导这种插入。这拟议研究的目的是确定哪些特征完整膜的一级和二级结构蛋白质使其能够插入磷脂双层中。这将是使用模型研究自发插入细菌视紫红质片段及其突变体进行二肉豆蔻酰磷脂酰胆碱的单层囊泡。细菌视紫红质，具有研究最充分的结构任何膜蛋白，由七个交叉的螺旋结构域组成来回穿过由以下连接的膜双层不同长度的亲水序列。初步研究有研究表明，当蛋白质被胰凝乳蛋白酶切割时在域 2 和 3 之间，产生的较大片段是仍然能够插入膜。附加片段含有结构域及其突变体的各种组合现在将通过蛋白水解和操纵产生使用位点特异性将基因克隆到表达载体中诱变。使用这些多肽的掺入实验磷脂囊泡将用于缩小范围细菌视紫红质中的序列对于插入磷脂双层。这些结果实验将构成使用额外的研究的基础突变以确定介导膜的结构插入并决定膜中的方向。最后，序列被鉴定为介导插入将测试整合膜蛋白以确定是否它们还可以锚定或移位其他亲水性蛋白质附着在它们上面。