外膜蛋白是一類非常獨特但又非常重要的蛋白質,他們廣泛的存在于原核生物的細胞外膜和真核生物的細胞器外膜中。結構上,它們都具有β-桶狀結構,不同的外膜蛋白的β-桶由不同偶數個β-折疊片組成,從8個到22個不等;功能上,它們具有為外膜提供通透性,維持外膜結構穩(wěn)定的作用。折疊好的外膜蛋白具有極強的穩(wěn)定性,常溫下,在2%的強離子型表面活性劑SDS中,外膜蛋白仍然具有正常的折疊結構;而在此溫度下,其他可溶蛋白在0.2%的SDS中就可以迅速失去結構。因此通常將加樣前是否對樣品進行95C加熱去折疊的SDS-PAGE稱為denatured SDS-PAGE和semi-native SDS-PAGE,并被用來研究外膜蛋白的折疊狀況。 外膜蛋白的研究經歷了漫長而又曲折的過程。起初人們并沒有意識到外膜蛋白的重要性,雖然有一些關于外膜脂多糖生成障礙與熱致死相關的文獻報道,但是人們還是把更多的注意力集中到膜間質蛋白的生成上,或者模糊的認為膜間質蛋白的生成就是外膜的生成,或者認為膜間質蛋白的錯誤折疊是引起細胞在脅迫條件下死亡的主要原因。隨著認識的深入,Ried, G.et al.發(fā)現與耐熱相關的脂多糖實際上幫助了外膜蛋白的正確生成,Mecsas, J.et al.通過遺傳篩選證明只有外膜蛋白的過表達才會對細胞產生來自胞外的蛋白質生成壓力,激活主要膜間質脅迫信號途徑σE通路。進一步的證據表明,膜間質的主要分子伴侶SurA和Skp都參與了外膜蛋白的正常生成,過表達膜間質分子伴侶可以補救脂多糖合成缺陷的表型,挽救外膜蛋白的異常生成。此后,Rizzitello, A.E.et al.和Sklar, J.G.et al.分別發(fā)現還發(fā)現膜間質的分子伴侶SurA與DegP和Skp存在平行結構的協(xié)作關系,surA和skp,surA和degP的雙敲除都會表現出致死表型并影響外膜蛋白的正常生成。整個認識過程中最重要的發(fā)現出現在2003年,Voulhoux, R.et al.發(fā)現,一種特殊的外膜蛋白Omp85,能夠捕捉膜間質中的自內膜轉運而來的其他外膜蛋白中間體,并引導它們進入外膜,它的敲除株會使外膜蛋白的生成出現嚴重的障礙并導致細菌的死亡,這一發(fā)現使得外膜蛋白生成這一鮮有問津的生物學領域真正引起了學術界的高度重視。這之后,Wu, T.et al.發(fā)現Omp85/YaeT/BamA這一因子是通過與YfgL (BamB), NlpB (BamC), YfiO (BamD)和SmpA (BamE)形成巨大的復合物(>230kDa)來發(fā)揮作用。最近,Krojer, T.et al.發(fā)現膜間質中的另一個與耐熱相關的蛋白DegP/HtrA,能夠和外膜蛋白形成穩(wěn)定的復合物DegP12/24-OMP,雖然其形成的機制尚不清楚,但是這一發(fā)現仍然說明膜間質中的脅迫更多是由于外膜蛋白的異常生成造成的。 最近我們實驗室通過生化和遺傳學的方法證明,主要外膜蛋白都存在穩(wěn)定的折疊中間態(tài),并且這些折疊中間態(tài)的存在是革蘭氏陰性菌能在脅迫條件下能正常存活的基礎條件。耐熱相關蛋白DegP/HtrA通過在Skp和SurA下游識別并分選這種折疊中間狀態(tài)來使細菌抵抗環(huán)境脅迫并為其提供致病性。DegP的分子伴侶活性和蛋白酶活性在分選外膜蛋白折疊中間狀態(tài)的過程中同時發(fā)揮著重要的作用,分子伴侶活性負責捕捉和穩(wěn)定不同折疊狀態(tài)的外膜蛋白,幫助部分折疊的外膜蛋白插膜,防止錯誤折疊的外膜蛋白吸附到外膜上;蛋白酶活性對于清除錯誤折疊的外膜蛋白至關重要。 Burgess, N.K., Dao, T.P., Stanley, A.M., & Fleming, K.G., Beta-barrel proteins that reside in theEscherichia coliouter membrane in vivo demonstrate varied folding behavior in vitro.J. Biol. Chem.283 (39), 26748-26758 (2008).
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