Methods for Studying the Interaction of Barnase with Its Inhibitor Barstar

  • Gideon Schreiber
Part of the Methods in Molecular Biology™ book series (MIMB, volume 160)


Barnase is a 110-residue extracellular ribonuclease produced by Bacillus amyloliquefaciens. The same organism produces an 89-residue polypeptide, barstar, which binds tightly to barnase and inhibits its potentially lethal RNase activity inside the cell. The three-dimentional structures of barnase and barstar are known both from X-ray crystallography and NMR spectroscopy (1, 2, 3, 4). The crystal structure of the complex of barnase with the barstar (C40A/C82A) double mutant has been solved by Guillet et al. (1993) 6 at 2.6 Å resolution and by Buckle et al. (1994) 5 at 2.0 Å resolution (5,5). Both barnase and barstar have proven to be excellent models for protein stability and folding studies (7, 8, 9, 10, 11). The two proteins form a very tight complex, with K d=10−14 M, half-life of about 2 d and an association rate constant of 4×108 M −1/s. The kinetic and thermodynamics of the barnase-barstar interaction were investigated in detail (12, 13, 14, 15, 16, 17, 18, 19, 20). High affinity is maintained at pH 7.0–9.0, while lowering the pH to under 4.5 eliminates binding completely. The plentiful structural and thermodynamic data available for this interaction, the ease of genetic manipulation and expression, and the excellent in vivo selection methods have made this interaction a target for many genetic selection techniques. Because free barnase is toxic, organisms expressing it can survive only if barstar is efficiently coexpressed. This positive selection system has been used as a convenient tool for transcriptional studies in plants. For example, the tissue specificity of the TA56 promoter was mapped by expressing a TA56/barnase fusion gene in tobacco plants and determining which tissues showed a requirement for barstar production (21).Using a similar approach, a transgenic maize plant was developed where a plant with the barnase gene is pollen-sterile, whereas a plant with the barnase and barstar gene is pollen-fertile.


Isothermal Titration Calorimetry Association Rate Constant Transgenic Maize Plant Barnase Gene TA56 Promoter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Bycroft, M., Ludvigs, S., Fersht, A. R., and Poulsen, F. M. (1991) Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spec-troscopy. Biochemistry 30, 8697–8701.PubMedCrossRefGoogle Scholar
  2. 2.
    Lubienski, M. J., Bycroft, M., Freund, S. M., and Fersht, A. R. (1994) Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy. Biochemistry 33, 8866–8877.PubMedCrossRefGoogle Scholar
  3. 3.
    Mauguen, Y., Hartley, R. W., Dodson, E. J., Dodson, G. G., Bricogne, G., Chothia, C., and Jack, A. (1982) Molecular structure of a new family of ribonucleases. Nature 29, 162–164.CrossRefGoogle Scholar
  4. 4.
    Ratnaparkhi, G. S., Ramachandran, S., Udgaonkar, J. B., and Varadarajan, R. (1998) Discrepancies between the NMR and X-ray structures of uncomplexed barstar: analysis suggests that packing densities of protein structures determined by NMR are unreliable. Biochemistry 37, 6958–6966.PubMedCrossRefGoogle Scholar
  5. 5.
    Buckle, A. M., Schreiber, G., and Fersht, A. R. (1994) Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. Biochemistry 33, 8878–8889.PubMedCrossRefGoogle Scholar
  6. 6.
    Guillet, V., Lapthorn, A., Hartley, R. W., and Maugen, Y. (1993) Recognition between a bacterial ribonuclease, barnase and its natural inhibitor, barstar. Structure 1, 165–177.PubMedCrossRefGoogle Scholar
  7. 7.
    Fersht, A. R. (1993) Protein folding and stability: the pathway of folding of barnase. FEBS Lett 325, 5–16.PubMedCrossRefGoogle Scholar
  8. 8.
    Fersht, A. R., Matouschek, A., and Serrano, L. (1992) The folding of an enzyme i: theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782.PubMedCrossRefGoogle Scholar
  9. 9.
    Nath, U., Agashe, V. R., and Udgaonkar, J. B. (1996) Initial loss of secondary structure in the unfolding of barstar [letter]. Nature Struct. Biol. 3, 920–923.PubMedCrossRefGoogle Scholar
  10. 10.
    Nolting, B., Golbik, R., Neira, J. L., Soler, G. A., Schreiber, G., and Fersht, A. R. (1997) The folding pathway of a protein at high resolution from microseconds to seconds. Proc. Natl. Acad. Sci. USA 94, 826–830.PubMedCrossRefGoogle Scholar
  11. 11.
    Zaidi, F. N., Nath, U., and Udgaonkar, J. B. (1997) Multiple intermediates and transition states during protein unfolding. Nature Struct. Biol. 4, 1016–1024.PubMedCrossRefGoogle Scholar
  12. 12.
    Frisch, C., Schreiber, G., Johnson, C. M., and Fersht, A. R. (1997) Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation. J. Mol. Biol. 267, 696–706.PubMedCrossRefGoogle Scholar
  13. 13.
    Hartley, R. W. (1993) Directed mutagenesis and barnase-barstar recognition. Biochemistry 32, 5978–5984.PubMedCrossRefGoogle Scholar
  14. 14.
    Martinez, J. C., Filimonov, V. V., Mateo, P. L., Schreiber, G., and Fersht, A. R. (1995) A calorimetric study of the thermal stability of barstar and its interaction with barnase. Biochemistry 34, 5224–5233.PubMedCrossRefGoogle Scholar
  15. 15.
    Schreiber, G., Buckle, A. M., and Fersht, A. R. (1994) Stability and function: two constraints in the evolution of barstar and other proteins. Structure 2, 945–951.PubMedCrossRefGoogle Scholar
  16. 16.
    Schreiber, G. and Fersht, A. R. (1993) Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. Biochemistry 32, 5145–5150.PubMedCrossRefGoogle Scholar
  17. 17.
    Schreiber, G. and Fersht, A. R. (1995) Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J. Mol. Biol. 248, 478–486.PubMedGoogle Scholar
  18. 18.
    Schreiber, G. and Fersht, A. R. (1996) Rapid, electrostatically assisted association of proteins. Nature Struct. Biol. 3, 427–431.PubMedCrossRefGoogle Scholar
  19. 19.
    Schreiber, G., Frisch, C., and Fersht, A. R. (1997) The role of Glu73 of barnase in catalysis and the binding of barstar. J. Mol. Biol. 270, 111–122.PubMedCrossRefGoogle Scholar
  20. 20.
    Yakovlev, G. I., Moiseyev, G. P., Protasevich, I. I., Ranjbar, B., Bocharov, A. L., Kirpichnikov, M. P., Gilli, R. M., et al. (1995) Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase. FEBS Lett. 366, 156–158.PubMedCrossRefGoogle Scholar
  21. 21.
    Beals, T. P. and Goldberg, R. B. (1997) A novel cell ablation strategy blocks tobacco anther dehiscence. Plant Cell. 9, 1527–1545.PubMedCrossRefGoogle Scholar
  22. 22.
    Mariani, C., Gossele, V., De Beuckeleer, M., De, B. M., Goldberg, R. B., De Greef, W., and Leemans, J. (1992) A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357, 384–387.CrossRefGoogle Scholar
  23. 23.
    Jucovic, M. and Hartley, R. W. (1996) Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface. Proc. Natl. Acad. Sci. USA 93, 2343–2347.PubMedCrossRefGoogle Scholar
  24. 24.
    Deyev, S. M., Yazynin, S. A., Kuznetsov, D. A., Jukovich, M., and Hartley, R. W. (1998) Ribonuclease-charged vector for facile direct cloning with positive selection. Mol. Gen. Genet. 259, 379–382.PubMedCrossRefGoogle Scholar
  25. 25.
    Buckle, A. M. and Fersht, A. R. (1994) Subsite binding in an RNase: structure of a barnase-tetranucleotide complex at 1.76 Å resolution. Biochemistry 33, 1644–1653.PubMedCrossRefGoogle Scholar
  26. 26.
    Guillet, V., Lapthorn, A., and Mauguen, Y. (1993) Three-dimensional structure of a barnase-3′GMP complex at 2.2A resolution. FEBS Lett. 330, 137–140.PubMedCrossRefGoogle Scholar
  27. 27.
    Wong, K. B., Fersht, A. R., and Freund, S. M. (1997) NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A. J. Mol. Biol. 268, 494–511.PubMedCrossRefGoogle Scholar
  28. 28.
    Bogan, A. A. and Thorn, K. S. (1998) Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Clackson, T. and Wells, J. A. (1995) A hot spot of binding energy in a hormone-receptor interface. Science 267, 383–386.PubMedCrossRefGoogle Scholar
  30. 30.
    Soler-Gonzalez, A. S. and Fersht, A. R. (1997) Helix stability in barstar peptides. Eur. J. Biochem. 249, 724–732.PubMedCrossRefGoogle Scholar
  31. 31.
    Kobe, B. and Deisenhofer, J. (1995) A structural basis of the interactions between leu-cine-rich repeats and protein ligands. Nature 374, 183–186.PubMedCrossRefGoogle Scholar
  32. 32.
    Gabdoulline, R. R. and Wade, R. C. (1997) Simulation of the diffusional association of barnase and barstar. Biophys. J. 72, 1917–1929.PubMedCrossRefGoogle Scholar
  33. 33.
    Janin, J. (1997) The kinetics of protein-protein recognition. Proteins 28, 153–161.PubMedCrossRefGoogle Scholar
  34. 34.
    Selzer, T. and Schreiber, G. (1999) Predicting the rate enhancement of protein complex formation from the electrostatic energy of interaction. J. Mol. Biol. 287, 409–419.PubMedCrossRefGoogle Scholar
  35. 35.
    Vijayakumar, M., Wong, K. Y., Schreiber, G., Fersht, A. R., Szabo, A., and Zhou, H. X. (1998) Electrostatic enhancement of diffusion-controlled protein-protein association: comparison of theory and experiment on barnase and barstar. J. Mol. Biol. 278, 1015–1024.PubMedCrossRefGoogle Scholar
  36. 36.
    Makarov, A. A., Protasevich, I. I., Lobachov, V. M., Kirpichnikov, M. P., Yakovlev, G. I., Gilli, R. M., et al. (1994) Thermostability of the barnase-barstar complex. FEBS Lett. 354, 251–254.PubMedCrossRefGoogle Scholar
  37. 37.
    Covell, D. G. and Wallqvist, A. (1997) Analysis of protein-protein interactions and the effects of amino acid mutations on their energetics. The importance of water molecules in the binding epitope. J. Mol. Biol. 269, 281–297.PubMedCrossRefGoogle Scholar
  38. 38.
    Paddon, C. J. and Hartley, R. W. (1987) Expression of bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. Gene 53, 11–19.PubMedCrossRefGoogle Scholar
  39. 39.
    Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.PubMedCrossRefGoogle Scholar
  40. 40.
    Serpesu, E. H., Shortle, D., and Mildvan, A. S. (1986) Kinetic and magnetic resonance studies of effects of genetic substitutions of a Ca++ liganding amino acid in Staphysococcal Nuclease. Biochemistry 25, 67–87.Google Scholar
  41. 41.
    Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.PubMedCrossRefGoogle Scholar
  42. 42.
    Mossakowska, D. E., Nyberg, K., and Fersht, A. R. (1989) Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry 28, 3843–3850.PubMedCrossRefGoogle Scholar
  43. 43.
    Day, A. G., Parsonage, D., Ebel, S., Brown, T., and Fersht, A. R. (1992) Barnase has subsites that give rise to large rate enhancements. Biochemistry 31, 6390–6395.PubMedCrossRefGoogle Scholar
  44. 44.
    Fersht, A. R. (1985) Enzyme Structure and Mechanism, W. H. Freeman, New York.Google Scholar
  45. 45.
    Horovitz, A., Serrano, L., Avron, B., Bycroft, M., and Fersht, A. R. (1990) Strength and cooperativity of contributions of surface salt bridges to protein stability. J. Mol. Biol. 216, 1031–1044.PubMedCrossRefGoogle Scholar
  46. 46.
    Frisch, C., Schreiber, G., and Fersht, A. R. (1995) Characterization of in vitro oxidized barstar. FEBS Lett. 370, 273–277.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Gideon Schreiber
    • 1
  1. 1.Department of Biological ChemistryWeizmann Institute of ScienceRehovotIsrael

Personalised recommendations