在非氫鍵鍵結位置上離胺酸側鏈長度對β-Hairpin穩定度的影響及在Rev 胜肽N端利用氫鍵代替物對於核糖核酸辨認的影響

許多因素對於蛋白質折疊與結構的穩定占有一定的影響力: 胺基酸本身的偏好、側鏈之間離子對作用力、疏水性效應、氫鍵、凡德瓦力。在本研究中, 我們對於 β-髮夾裡非氫鍵鍵結位置上離胺酸側鏈長度對於在摺板偏好的效應做了研究。β-髮夾胜肽HPTAlaXaa (Xaa = Dap, Dab, Orn, Lys,為離胺酸同系物)被設計將9號位置離胺酸的側鏈做一系列的變短。胜肽利用固相胜肽合成並純化至95%純度, 並利用二維核磁共振光譜做分析。β-髮夾的結構利用化學位移差異、3JHNα 偶合常數和 NOE 訊號作確認。利用與全折疊與全不折疊的參考胜肽的化學位移的差異, 胜肽折疊百分率和其自由能變化可以被推導出來。HPTAlaXaa 離胺酸系列的胜肽折疊率為: HPTAlaDap ~ HPTAlaDab < HPTAlaOrn ~ HPTAlaLys, 顯示在 β-髮夾胜肽含有離胺酸同系物的側鏈越長, β-髮夾結構越穩定。與HIV相關的Rev蛋白與RRE RNA結合後, 會轉譯後修飾調控未經修剪和修剪過的mRNA從核內到細胞質。Rev胜肽是random coil的結構, 但是一旦與RRE RNA做結合後會轉變成α-螺旋的結構。氫鍵替代物是許多種用於穩定α-螺旋的跨連結系統中的一種, 利用共價鍵C=C-C-N取代一般常見的(i, i+4) C=O…C-N氫鍵來穩定短的α-螺旋。為了要合成含有氫鍵代替物的胜肽, 對於含有N-allyl的二胜肽的合成策略作了設計與修改。兩條自然界Rev胜肽利用固態胜肽方式合成, 利用圓二色光譜鑑定其二級結構為random coil. 利用電泳偏移分析Rev胜肽對於RRE RNA的結合專一性, 結果顯示解離常數與之前的文獻是相似的。

關鍵字

離胺酸；摺板偏好； Rev ； RRE RNA ；氫鍵代替物

並列摘要

There are many factors that contribute to protein folding and structure stability: intrinsic propensity of amino acids, side chain ion pairing interaction, hydrophobic effect, hydrogen bonding, van der Waals interaction. In this study, we focused on the effect of lysine side chain length on sheet propensity at a non-hydrogen bonded strand position in β-hairpin. The β-hairpin peptides HPTAlaXaa (Xaa= Dap, Dab, Orn, Lys) were designed with the side chain of Lys9 systematically shortened to investigate the effect of Lys side chain length on sheet propensity. The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. All peptides were purified to 95% purity and were analyzed by 2D NMR experiments. Sequence specific assignment was performed. The hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants,and NOE signals.The fraction folded and ΔG of peptides were derived by comparing the chemical shifts with the fully folded and unfolded reference peptides. The percent folding of HPTAlaXaa peptides with Lys analogs at the guest position followed the trend: HPTAlaDap ~ HPTAlaDab < HPTAlaOrn ~ HPTAlaLys, showing that the longer the Lys analogue side chain, the more stable the β-haiprin structure. The HIV Rev protein binds RRE RNA to regulate the transport of unspliced and spliced mRNA from the nucleus to the cytoplasm posttranscriptionally. The Rev peptide is a random-coil. However, the conformation of the Rev peptide changes to an α-helix while binding to RRE RNA. Hydrogen bond surrogate (HBS) is one of the several cross-linking systems for stabilizing an α-helix, using the covalent bond C=C-C-N to substitute the C=O…H-N (i, i+4) hydrogen bond in a short helix. In order to synthesize an HBS peptide, strategy for synthesis of dipeptides that contained an allyl group on the amino group was designed and refined. Two wild type Rev peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. The secondary structure of the two peptides was random-coil analyzed by circular dichroism spectroscopy. The binding specificity of the Rev peptides was determined by gel shift assay. The dissociation constants of the Rev peptides were similar to previous studies.

並列關鍵字

Lys ； sheet propensity ； β-hairpin ； Rev ； RRE RNA ； hydrogen bond surrogate.

參考文獻

6. Cheng, R. P.; Wang, W. R.; Girinath, P.; Yang, P. A.; Ahmad, R.; Li, J. H.; Hart, P.; Kokona, B.; Fairman, R.; Kilpatrick, C.; Argiros, A. Effect of glutamate side

5. Cheng, R. P.; Girinath, P.; Suzuki, Y.; Kuo, H. T.; Hsu, H. C.; Wang, W. R.; Yang, P. A.; Gullickson, D.; Wu, C. H.; Koyack, M. J.; Chiu, H. P.; Weng, Y. J.;

16. Joosten, R. P.; Beek, T. A. H. T.; Krieger, E.; Hekkelman, M. L.; Hooft, R. W.

1. Crick, F. Central dogma of molecular biology. Nature 1970, 227, 561.

3. Fersht, A. R. Structure and mechanism in protein science. A guide to enzyme catalysis and protein folding. W. H. Freeman and Co.: New York, 1999.

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在非氫鍵鍵結位置上離胺酸側鏈長度對β-Hairpin穩定度的影響及在Rev 胜肽N端利用氫鍵代替物對於核糖核酸辨認的影響

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