title: The STINT-NMR method for studying in-cell protein-protein interactions.
abstract: This unit describes critical components and considerations required to study protein-protein structural interactions inside a living cell by using NMR spectroscopy (STINT-NMR). STINT-NMR entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at the level of single amino acid residues. The advantages and limitations of STINT-NMR are discussed, along with the differences between studying macromolecular interactions in vitro and in vivo (in-cell). Also described are considerations in the design of STINT-NMR experiments, focusing on selecting appropriate overexpression plasmid vectors, sample requirements and instrumentation, and the analysis of STINT-NMR data, with specific examples drawn from published works. Applications of STINT-NMR, including an in-cell methodology to post-translationally modify interactor proteins and an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) are presented.
authors.affiliation: State University of New York at Albany, Albany, New York.
authors.forename: David S
authors.initials: DS
authors.lastname: Burz
authors.affiliation: State University of New York at Albany, Albany, New York.
authors.forename: Alexander
authors.initials: A
authors.lastname: Shekhtman
mesh_terms: D003585:Cell Biology; D055808:Drug Discovery; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics
references.citation: Bader, G.D., Betel, D., and Hogue, C.W. 2003. BIND: The Biomolecular Interaction Network Database. Nucleic Acids Res. 31:248-250.
references.citation: Burz, D.S. and Shekhtman, A. 2008. In-cell biochemistry using NMR spectroscopy. PLoS ONE 3:e2571.
references.citation: Burz, D.S., and Shekhtman, A. 2009. Structural biology: Inside the living cell. Nature 458:37-38.
references.citation: Burz, D.S., Dutta, K., Cowburn, D., and Shekhtman, A. 2006a. In-cell NMR for protein-protein interactions (STINT-NMR). Nat. Protoc. 1:146-152.
references.citation: Burz, D.S., Dutta, K., Cowburn, D., and Shekhtman, A. 2006b. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nat. Methods 3:91-93.
references.citation: Clague, M.J. and Urbe, S. 2001. The interface of receptor trafficking and signalling. J. Cell Sci. 114:3075-3081.
references.citation: Cruzeiro-Silva, C., Albernaz, F.P., Valente, A.P., and Almeida, F.C. 2006. In-Cell NMR spectroscopy: Inhibition of autologous protein expression reduces Escherichia coli lysis. Cell Biochem. Biophys. 44:497-502.
references.citation: Fernandes, P.B. 1998. Technological advances in high-throughput screening. Curr. Opin. Chem. Biol. 2:597-603.
references.citation: Gardner, K.H. and Kay, L.E. 1998. The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu. Rev. Biophys. Biomol. Struct. 27:357-406.
references.citation: Gerstein, M. 2000. Integrative database analysis in structural genomics. Nat. Struct. Biol. 7:960-963.
references.citation: Giepmans, B.N., Adams, S.R., Ellisman, M.H., and Tsien, R.Y. 2006. The fluorescent toolbox for assessing protein location and function. Science 312:217-224.
references.citation: Groll, M., Bochtler, M., Brandstetter, H., Clausen, T., and Huber, R. 2005. Molecular machines for protein degradation. Chembiochem 6:222-256.
references.citation: Haglund, K., Di Fiore, P.P., and Dikic, I. 2003. Distinct monoubiquitin signals in receptor endocytosis. Trends Biochem. Sci. 28:598-603.
references.citation: Hajduk, P.J. and Burns, D.J. 2002. Integration of NMR and high-throughput screening. Comb. Chem. High Throughput Screen. 5:613-621.
references.citation: Heitman, J., Movva, N.R., and Hall, M.N. 1991. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905-909.
references.citation: Inomata, K., Ohno, A., Tochio, H., Isogai, S., Tenno, T., Nakase, I., Takeuchi, T., Futaki, S., Ito, Y., Hiroaki, H., and Shirakawa, M. 2009. High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Nature 458:106-109.
references.citation: Jansen, R., Greenbaum, D., and Gerstein, M. 2002. Relating whole-genome expression data with protein-protein interactions. Genome Res. 12:37-46.
references.citation: Krogan, N.J., Cagney, G., Yu, H., Zhong, G., Guo, X., Ignatchenko, A., Li, J., Pu, S., Datta, N., Tikuisis, A.P., Punna, T., Peregrín-Alvarez, J.M., Shales, M., Zhang, X., Davey, M., Robinson, M.D., Paccanaro, A., Bray, J.E., Sheung, A., Beattie, B., Richards, D.P., Canadien, V., Lalev, A., Mena, F., Wong, P., Starostine, A., Canete, M.M., Vlasblom, J., Wu, S., Orsi, C., Collins, S.R., Chandran, S., Haw, R., Rilstone, J.J., Gandi, K., Thompson, N.J., Musso, G., St Onge, P., Ghanny, S., Lam, M.H., Butland, G., Altaf-Ul, A.M., Kanaya, S., Shilatifard, A., O'Shea, E., Weissman, J.S., Ingles, C.J., Hughes, T.R., Parkinson, J., Gerstein, M., Wodak, S.J., Emili, A., and Greenblatt, J.F. 2006. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637-643.
references.citation: Li, C., Charlton, L.M., Lakkavaram, A., Seagle, C., Wang, G., Young, G.B., Macdonald, J.M., and Pielak, G.J. 2008. Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy. J. Am. Chem. Soc. 130:6310-6311.
references.citation: Marmor, M.D. and Yarden, Y. 2004. Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 23:2057-2070.
references.citation: Pellecchia, M., Sem, D.S., and Wuthrich, K. 2002. NMR in drug discovery. Nat. Rev. Drug Discov. 1:211-219.
references.citation: Pellecchia, M., Bertini, I., Cowburn, D., Dalvit, C., Giralt, E., Jahnke, W., James, T.L., Homans, S.W., Kessler, H., Luchinat, C., Meyer, B., Oschkinat, H., Peng, J., Schwalbe, H., and Siegal, G. 2008. Perspectives on NMR in drug discovery: A technique comes of age. Nat. Rev. Drug Discov. 7:738-745.
references.citation: Peng, J.W., Lepre, C.A., Fejzo, J., Abdul-Manan, N., and Moore, J.M. 2001. Nuclear magnetic resonance-based approaches for lead generation in drug discovery. Methods Enzymol. 338:202-230.
references.citation: Piotto, M., Saudek, V., and Sklenar, V. 1992. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2:661-665.
references.citation: Rain, J.C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S., Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemama, Y., Labigne, A., and Legrain, P. 2001. The protein-protein interaction map of Helicobacter pylori. Nature 409:211-215.
references.citation: Sakakibara, D., Sasaki, A., Ikeya, T., Hamatsu, J., Hanashima, T., Mishima, M., Yoshimasu, M., Hayashi, N., Mikawa, T., Walchli, M., Smith, B.O., Shirakawa, M., Guntert, P., and Ito, Y. 2009. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458:102-105.
references.citation: Sali, A., Glaeser, R., Earnest, T., and Baumeister, W. 2003. From words to literature in structural proteomics. Nature 422:216-225.
references.citation: Schuffenhauer, A., Ruedisser, S., Marzinzik, A.L., Jahnke, W., Blommers, M., Selzer, P., and Jacoby, E. 2005. Library design for fragment based screening. Curr. Top. Med. Chem. 5:751-762.
references.citation: Seet, B.T., Dikic, I., Zhou, M.M., and Pawson, T. 2006. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7:473-483.
references.citation: Selenko, P., Serber, Z., Gadea, B., Ruderman, J., and Wagner, G. 2006. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Proc. Natl. Acad. Sci. U.S.A. 103:11904-11909.
references.citation: Serber, Z. and Dotsch, V. 2001. In-cell NMR spectroscopy. Biochemistry 40:14317-14323.
references.citation: Shuker, S.B., Hajduk, P.J., Meadows, R.P., and Fesik, S.W. 1996. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531-1534.
references.citation: Silverman, L., Campbell, R., and Broach, J.R. 1998. New assay technologies for high-throughput screening. Curr. Opin. Chem. Biol. 2:397-403.
references.citation: Slade, K.M., Baker, R., Chua, M., Thompson, N.L., and Pielak, G.J. 2009. Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli. Biochemistry 48:5083-5089.
references.citation: Tugarinov, V., Hwang, P.M., and Kay, L.E. 2004. Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Annu. Rev. Biochem. 73:107-146.
references.citation: Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G. Yang, M., Johnston, M., Fields, S., and Rothberg, J.M. 2000. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623-627.
references.citation: Xie, J., Reverdatto, S., Frolov, A., Hoffmann, R., Burz, D.S., and Shekhtman, A. 2008. Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE). J. Biol. Chem. 283:27255-27269.
references.citation: Xie, J., Thapa, R., Reverdatto, S., Burz, D.S., and Shekhtman, A. 2009. Screening of small molecule interactor library by using in-cell NMR spectroscopy (SMILI-NMR). J. Med. Chem. 52:3516-3522.
references.citation: Zhu, Z., Pilpel, Y., and Church, G.M. 2002. Computational identification of transcription factor binding sites via a transcription-factor-centric clustering (TFCC) algorithm. J. Mol. Biol. 318:71-81.
references.citation: Zuiderweg, E.R. 2002. Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41:1-7.