优化的Native PAGE蛋白回收方法在AFM中的应用
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摘要
原子力显微镜(AFM)可以在生理条件下获得生物大分子的自然结构信息而不需要进行图像处理。虽然对含有大小范围不一的异质样品成像可以得到几纳米的分辨率,但是具有更高的亚纳米级分辨率的图像仅可以从含有单一亚基数目的高度均一的蛋白复合物获得。在重组蛋白的制备中这种蛋白复合物经常不存在,尤其是对于膜蛋白则更具有挑战性。本研究中,研究者以一种熟知的膜成孔蛋白复合物Perfringolysin O(PFO)作为模型系统,研制出一种方法来分离含有单一亚基数目的复合物,并用于随后在液相的AFM成像。具体而言,研究人员通过非变性聚丙烯酰胺凝胶电泳(Native PAGE)分离出该复合物中特定大小的蛋白成分,然后提取时采用一种新方法使结构完整保持。预计这种方法不仅对更多其他蛋白的高分辨率AFM研究有效用,而且在基于单分子方法和生物技术应用的研究上有更普遍的用途。
Atomic force microscopy(AFM) can determine structural information of biological macromolecules under physiological conditions directly from unprocessed images. However, while images with a resolution of a few nanometers can be obtained from heterogeneous samples containing macromolecules with a range of sizes and stoichiometries, higher resolution images with sub-nanometer-scale details have only been obtained from highly homogeneous protein complexes of a single stoichiometry, which is frequently not present in preparations of recombinant proteins and is especially challenging with membrane proteins. Here, using perfringolysin O(PFO), a well-studied membrane pore-forming protein that is well known to form a wide range of stoichiometries, as a model system, we had developed a strategy to isolate a single-sized complex that was suitable for subsequent AFM imaging under solution. In particular, we isolated a specific size of this complex using native polyacrylamide gel electrophoresis(Native PAGE), followed by a structure-preserving method to extract the isolated complex. We anticipated that this method would prove useful not only for high resolution AFM studies of many other proteins,but also, more generally, for studies using other single-molecule based methods and biotechnological applications.
Atomic force microscopy(AFM) can determine structural information of biological macromolecules under physiological conditions directly from unprocessed images. However, while images with a resolution of a few nanometers can be obtained from heterogeneous samples containing macromolecules with a range of sizes and stoichiometries, higher resolution images with sub-nanometer-scale details have only been obtained from highly homogeneous protein complexes of a single stoichiometry, which is frequently not present in preparations of recombinant proteins and is especially challenging with membrane proteins. Here, using perfringolysin O(PFO), a well-studied membrane pore-forming protein that is well known to form a wide range of stoichiometries, as a model system, we had developed a strategy to isolate a single-sized complex that was suitable for subsequent AFM imaging under solution. In particular, we isolated a specific size of this complex using native polyacrylamide gel electrophoresis(Native PAGE), followed by a structure-preserving method to extract the isolated complex. We anticipated that this method would prove useful not only for high resolution AFM studies of many other proteins,but also, more generally, for studies using other single-molecule based methods and biotechnological applications.
引文
Ad amcik J.,Berquand A.,and Mezzenga R.,2011,Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy,Appl.Phys.Lett.,98(19):193701
Braha O.,Walker B.,Cheley S.,Kasianowicz J.J.,Song L.,Gouaux J.E.,and Bayley H.,1997,Designed protein pores as components for biosensors,Chemical and Biology,4(7):497-505
Czajkowsky D.M.,Hotze E.M.,Shao Z.F.,and Tweten R.K.,2004,Vertical collapse of a cytolysinprepore moves its transmembrane beta-hairpins to the membrane,The EMBO Journal,23(16):3206-3215
Czajkowsky D.M.,and Shao Z.F.,1998,Submolecular resolution of single macromolecules with atomic force microscopy,FEBSLetters,430:51-54
Czajkowsky D.M.,and Shao Z.F.,2009,The human Ig M pentamer is a mushroom-shaped molecule with a flexural bias,Proc.Natl.Acad.Sci.USA,106(35):14960-14965
Czajkowsky D.M.,Sun J.L.,and Shao Z.F.,2015,Single molecule compression reveals intra-protein forces drive cytotoxin pore formation,Elife,4:e08421
Fukuma T.,Kimura M.,Kobayashi K.,Matsushige K.,and Yamada H.,2005,Development of low noise cantilever deflection sensor for multienvironment frequency-modulation atomic force microscopy,Review of Scientific Instruments,76(5):668-1314
Hansma P.K.,Cleveland J.P.,Radmacher M.,Walters D.A.,Hillner P.E.,BezanillaM.,Fritz M.,Vie D.,Hansma H.G.,Prater C.B.,Massie J.,Fukunaga L.,Gurley J.,and Elings V.,1994,Tapping mode atomic force microscopy in liquids,Appl.Phys.Lett.,64(13):1738-1740
Jin Y.,and Manabe T.,2005,High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry,Electrophoresis,26(6):1019-1028
Muller D.J.,Sass H.J.,Muller S.A.,Buldt G.,and Engel A.,1999,Surface structures of native bacteriorhodopsin depend on the molecular packing arrangement in the membrane,J.Mol.Biol.,285(5):1903-1909
Shao Z.F.,Mou J.X.,Czajkowsky D.M.,Yang J.,and Yuan J.Y.,1996,Biological atomic force microscopy:what is achieved and what is needed,Advances in Physics,45(1):1-86
Shepard L.A.,Heuck A.P.,Hamman B.D.,Rossjohn J.,Parker M.W.,Ryan K.R.,Johnson A.E.,and Tweten R.K.,1998,Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O:an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy,Biochemistry,37(41):14563-14574
Shepard L.A.,Shatursky O.,Johnson A.E.,and Tweten R.K.,2000,The mechanism of pore assembly for a cholesterol-dependent cytolysin:formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins,Biochemistry,39(33):10284-10293
Tweten R.K.,1988,Cloning and expression in Escherichia coli of the perfringolysin O(theta-toxin)gene from Clostridium perfringens and characterization of the gene product,Infection and Immunity,56(12):3228-3234
Tweten R.K.,Hotze E.M.,and Wade K.R.,2015,Theunique molecular choreography of giant pore formation by the cholesterol-dependent cytolysins of gram-positive bacteria,Annu.Rev.Microbiol.,69:323-340
Wittig I.,Braun H.P.,and Schagger H.,2006,Blue native PAGE,Nat.Protoc.,1(1):418-428
Braha O.,Walker B.,Cheley S.,Kasianowicz J.J.,Song L.,Gouaux J.E.,and Bayley H.,1997,Designed protein pores as components for biosensors,Chemical and Biology,4(7):497-505
Czajkowsky D.M.,Hotze E.M.,Shao Z.F.,and Tweten R.K.,2004,Vertical collapse of a cytolysinprepore moves its transmembrane beta-hairpins to the membrane,The EMBO Journal,23(16):3206-3215
Czajkowsky D.M.,and Shao Z.F.,1998,Submolecular resolution of single macromolecules with atomic force microscopy,FEBSLetters,430:51-54
Czajkowsky D.M.,and Shao Z.F.,2009,The human Ig M pentamer is a mushroom-shaped molecule with a flexural bias,Proc.Natl.Acad.Sci.USA,106(35):14960-14965
Czajkowsky D.M.,Sun J.L.,and Shao Z.F.,2015,Single molecule compression reveals intra-protein forces drive cytotoxin pore formation,Elife,4:e08421
Fukuma T.,Kimura M.,Kobayashi K.,Matsushige K.,and Yamada H.,2005,Development of low noise cantilever deflection sensor for multienvironment frequency-modulation atomic force microscopy,Review of Scientific Instruments,76(5):668-1314
Hansma P.K.,Cleveland J.P.,Radmacher M.,Walters D.A.,Hillner P.E.,BezanillaM.,Fritz M.,Vie D.,Hansma H.G.,Prater C.B.,Massie J.,Fukunaga L.,Gurley J.,and Elings V.,1994,Tapping mode atomic force microscopy in liquids,Appl.Phys.Lett.,64(13):1738-1740
Jin Y.,and Manabe T.,2005,High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry,Electrophoresis,26(6):1019-1028
Muller D.J.,Sass H.J.,Muller S.A.,Buldt G.,and Engel A.,1999,Surface structures of native bacteriorhodopsin depend on the molecular packing arrangement in the membrane,J.Mol.Biol.,285(5):1903-1909
Shao Z.F.,Mou J.X.,Czajkowsky D.M.,Yang J.,and Yuan J.Y.,1996,Biological atomic force microscopy:what is achieved and what is needed,Advances in Physics,45(1):1-86
Shepard L.A.,Heuck A.P.,Hamman B.D.,Rossjohn J.,Parker M.W.,Ryan K.R.,Johnson A.E.,and Tweten R.K.,1998,Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O:an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy,Biochemistry,37(41):14563-14574
Shepard L.A.,Shatursky O.,Johnson A.E.,and Tweten R.K.,2000,The mechanism of pore assembly for a cholesterol-dependent cytolysin:formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins,Biochemistry,39(33):10284-10293
Tweten R.K.,1988,Cloning and expression in Escherichia coli of the perfringolysin O(theta-toxin)gene from Clostridium perfringens and characterization of the gene product,Infection and Immunity,56(12):3228-3234
Tweten R.K.,Hotze E.M.,and Wade K.R.,2015,Theunique molecular choreography of giant pore formation by the cholesterol-dependent cytolysins of gram-positive bacteria,Annu.Rev.Microbiol.,69:323-340
Wittig I.,Braun H.P.,and Schagger H.,2006,Blue native PAGE,Nat.Protoc.,1(1):418-428