BINDING OF PRENYLATED AND POLYBASIC PEPTIDES TO MEMBRANES - AFFINITIES AND INTERVESICLE EXCHANGE
BINDING OF PRENYLATED AND POLYBASIC PEPTIDES TO MEMBRANES - AFFINITIES AND INTERVESICLE EXCHANGE
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DOI：
10.1021/bi00037a032
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发表时间：
1995-09-19
期刊：
影响因子：
2.9
通讯作者：
GELB, MH
中科院分区：
文献类型：
--
作者：
GHOMASHCHI, F;ZHANG, XH;GELB, MH
小的GTP结合蛋白G25K和蛋白K-RAS 4B含有戊烯基团(香叶基和法尼基)，这些基团是硫醚连接到C-末端的半胱氨酸，半胱氨酸在其α-羧基上甲基化。这些蛋白质和许多其他的戊烯基蛋白质一样，在它们的C末端附近也有一串碱性残基。根据人脑G25K和人K-RAS 4B的C-末端序列，合成了一系列预苯基化多肽，并对其膜结合亲和力进行了分析。含有N-末端N-乙酰色氨酸的G25K多肽可以通过荧光共振能量转移检测到它们与含有微量丹磺化磷脂的膜的结合。缺少戊烯基团和C-末端甲酯的G25K多肽不能与囊泡结合，如果是香叶基香叶化的G25K多肽，结合能力会增加500倍以上。对于法尼化的多肽，如果囊泡含有磷脂酰丝氨酸，C末端的甲基化至少使膜亲和力增加60倍，如果没有这种酸性脂质，则增加3倍。只有当囊泡含有磷脂酰丝氨酸时，香叶基香叶酰化和甲基化的G25K肽才会在几分钟内不可逆地附着在囊泡上，而相应的未甲基化或法尼化和甲基化的多肽会从中性或阴离子囊泡中迅速解离(不到几秒钟)。非甲基化K-RAS 4B肽的法尼斯化使其与含有酸性磷脂(磷脂酰甘油或磷脂酰丝氨酸)的囊泡的亲和力增加70倍，甲基化导致膜亲和力额外显著增加(150倍)。用较长的香叶基香叶基取代法尼基可使膜结合力提高8倍。香叶基香叶酰化K-RAS多肽的甲基化使其与酸性脂类囊泡的结合增加了100倍；而与纯磷脂酰胆碱囊泡(无净电荷)的结合只增加了3倍。磷脂酰胆碱囊泡中含有20%的酸性磷脂，与纯磷脂酰胆碱囊泡相比，法尼化和香叶酰化K-RAS 4B甲基化多肽的结合力分别提高了300倍和700倍。提出了一个简单的模型，以合理地解释C端戊烯基半胱氨酸甲酯与多碱多肽序列在戊烯基多肽与含有酸性磷脂的囊泡结合方面的协同效应。
The small GTP-binding protein G25K and the protein K-Ras 4B contain prenyl groups (geranylgeranyl and farnesyl, respectively) that are thioether linked to a C-terminal cysteine which is methylated on its alpha-carboxyl group. These proteins, like many other prenyl proteins, also have a string of basic residues near their C-termini. A series of prenylated peptides based on the C-terminal sequences of human brain G25K and human K-Ras 4B were synthesized and analyzed for their membrane binding affinities. G25K peptides containing an N-terminal N-acetyltryptophan group were studied because their binding to membranes containing a trace of dansylated phospholipid could be detected by fluorescence resonance energy transfer. The G25K peptide lacking a prenyl group and a C-terminal methyl ester did not detectably bind to vesicles, and binding was enhanced by more than 500-fold if the peptide was geranylgeranylated. For the farnesylated peptide, methylation of the C-terminus increased membrane affinity by at least 60-fold if the vesicles contained phosphatidylserine and by 3-fold if they lacked this acidic lipid. The geranylgeranylated and methylated G25K peptide remains irreversibly attached to vesicles over several minutes only if the vesicles contain phosphatidylserine, whereas the corresponding nonmethylated or farnesylated and methylated peptides dissociate rapidly (less than a few seconds) from neutral or anionic vesicles. Farnesylation of the nonmethylated K-Ras 4B peptide enhances its affinity to vesicles containing acidic phospholipids (phosphatidylglycerol or phosphatidylserine) by 70-fold, and methylation leads to an additional dramatic (150-fold) increase in membrane affinity. Replacement of the farnesyl group by the longer geranylgeranyl group enhances membrane binding by 8-fold. Methylation of the geranylgeranylated K-Ras peptide enhances its binding to vesicles with acidic lipids by 100-fold; the corresponding enhancement with pure phosphatidylcholine vesicles (no net charge) is only 3-fold. The presence of 20% acidic phospholipid in phosphatidylcholine vesicles enhances the binding of the farnesylated and geranylgeranylated K-Ras 4B methylated peptides by 300- and 700-fold, respectively, compared to pure phosphatidylcholine vesicles. A simple model is presented to rationalize the synergistic effects of a C-terminal prenylated cysteine methyl ester combined with a polybasic peptide sequence on the binding of prenylated peptides to vesicles containing acidic phospholipids.