Abstract
本文介绍的方法到感兴趣的蛋白质和其他因素之间的检测和分析动态相互作用的体内 。我们的方法是基于最初是由彼得·舒尔茨和他的同事开发了1琥珀抑制技术。琥珀突变首先引入在编码目的蛋白质的基因的一个特定的密码子。琥珀突变体，然后在大肠杆菌中表达大肠杆菌连同编码基因的琥珀抑制tRNA和从詹氏甲烷球菌来源的氨基酰-tRNA合成酶。使用这种系统中，光活化的氨基酸类似物对 - 苯甲酰基苯（BPA）被结合在琥珀密码子。细胞，然后用紫外光照射到BPA残余物共价连接到蛋白质，都设在3-8埃。光交联是在用脉冲追踪标记和感兴趣的蛋白质的免疫沉淀结合，以便监测变化进行这发生在几秒钟的时间尺度来分钟蛋白质 - 蛋白质相互作用。我们优化了程序，研究了由两个独立的结构域，其被集成到外膜，并且被转运到细胞外空间中的结构域的结构域的细菌毒力因子的组件，但该方法可用于研究许多不同装配过程和生物学途径在原核和真核细胞。原则上相互作用的因素，甚至特定的相互作用结合到感兴趣的蛋白质因子的残基可以通过质谱法来识别。
Materials
| Name | Company | Catalog Number | Comments |
| QuikChange II Site-Directed Mutagenesis Kit | Agilent | 200521 | |
| BPA (H-p-Bz-Phe-OH) | Bachem | F-2800 | |
| TRAN35S-LABEL, Metabolic Labeling Reagent (35S-L-methionine and 35S-L-cysteine, >1,000 Ci/mmol) | MP Biomedicals | 51006 | |
| Spectroline SB-100P Super-High-Intensity UV Lamp, 365 nm | Spectronics Corporation | SB-110P | |
| Spectroline Replacement Bulb 100S | Spectronics Corporation | 11-992-15 | |
| Falcon Tissue Culture Plate, 6-well | Becton Dickinson Labware | 353046 | |
| Disposable 125 ml Erlenmeyer flask | Corning | 430421 | |
| Innova 3100 shaking water bath | New Brunswick Scientific | n.a. |
References
- Wang, L., Xie, J., Schultz, P. G.
- von Mering, C., et al. Comparative assessment of large-scale data sets of protein-protein interactions. Nature. 417, 399-403 (2002).
- Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. PNAS. 99, 11020-11024 (2002).
- Farrell, I. S., Toroney, R., Hazen, J. L., Mehr, R. A., Chin, J. W. Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Nat. Methods. 2 (5), 377-384 (2005).
- Wittelsberger, A., Mierke, D. F., Rosenblatt, M. Mapping ligand-receptor interfaces: approaching the resolution limit of benzophenone-based photaffinity scanning. Chem. Biol. Drug Des. 71, 380-383 (2008).
- Ieva, R., Tian, P., Peterson, J. H., Bernstein, H. D. Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain. PNAS. 108, 383-391 (2011).
- Wang, F., Robbins, S., Guo, J., Shen, W., Schultz, P. G. Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis. PLoS One. 5, (2010).
- Deiters, A., et al. Adding amino acids with novel reactivity to the genetic code of Saccharyomycescerevisiae. J. Am. Chem. Soc. 125, 11782-11783 (2003).
- Young, T. S., Ahmad, I., Brock, A., Schultz, P. G. Expanding the genetic repertoire of the methylotrophic yeast Pichiapastoris. Biochemistry. 48 (12), 2643-2653 (2009).
- Hino, N., et al. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nat. Methods. 2 (3), 201-206 (2005).
- Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P. G. Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat. Methods. 4 (3), 239-244 (2007).
- Leyton, D. L., Rossiter, A. E., Henderson, I. R. From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis. Nat. Rev. Microbiol. 10 (3), 213-225 (2012).
- Ieva, R., Bernstein, H. D. Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane. PNAS. 106, 19120-19125 (2009).
- Pavlova, O., Peterson, J. H., Ieva, R., Bernstein, H. D. Mechanistic link between barrel assembly and the initiation of autotransporter secretion. PNAS. 110, (2013).
- Harlow, D., Lane, D. Using antibodies: a laboratory manual. , Cold Spring Harbor Laborator Press. Cold Spring Harbor, NY. (1999).
- Barnard, T. J., Dautin, N., Lukacik, N., Bernstein, P., Buchanan, S. K. Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat. Struct. Mol. Biol. 14 (12), 1214-1220 (2007).
- Akiyama, Y., Ito, K. SecY protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompT protease in vitro. Biochem. Biophys. Res. Commun. 167 (2), 711-715 (1990).
