Barnase Explained
| Barnase |
| Width: | 260px |
| Symbol: | Barnase |
| Pdb: | 1BRS |
| Pdb Supplemental: | More structures |
| Uniprot: | P00648 |
| Ecnumber: | 3.1.27.- |
Barnase (a portmanteau of "BActerial" "RiboNucleASE") is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. It is synthesized and secreted by the bacterium Bacillus amyloliquefaciens, but is lethal to the cell when expressed without its inhibitor barstar. The inhibitor binds to and occludes the ribonuclease active site, preventing barnase from damaging the cell's RNA after it has been synthesized but before it has been secreted. The barnase/barstar complex is noted for its extraordinarily tight protein-protein binding, with an on-rate of 108s−1M−1.
Protein folding studies
Barnase has no disulfide bonds, nor does it require divalent cations or non-peptide components to fold. This simplicity, in combination with its reversible folding transition, means that barnase has been extensively studied in order to understand how proteins fold.[1] [2] [3] The folding of barnase has been extensively studied in the laboratory of Alan Fersht, who used it as the test case in developing a method of characterizing protein folding transition states known as phi value analysis.
Active site and catalytic mechanism
Barnase catalyzes hydrolysis at diribonucleotide GpN sites. Cleavage occurs in two steps using a general acid-base mechanism: a cyclic intermediate is formed during the first transesterification step, which is then hydrolysed to release the cleaved RNA. The two most important residues involved in catalysis are Glu73 and His102, which are both essential for enzymatic activity. Glu73 is the general base whilst His102 is the general acid. Although it is not directly involved in acid-base catalysis, Lys27 is also critical for activity; it has been implicated in transition-state substrate binding.[4]
See also
Further reading
- Arcus VL, Vuilleumier S, Freund SM, Bycroft M, Fersht AR . Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state . Proceedings of the National Academy of Sciences of the United States of America . 91 . 20 . 9412–9416 . September 1994 . 7937780 . 44822 . 10.1073/pnas.91.20.9412 . free . 1994PNAS...91.9412A .
- Arcus VL, Vuilleumier S, Freund SM, Bycroft M, Fersht AR . A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding . Journal of Molecular Biology . 254 . 2 . 305–321 . November 1995 . 7490750 . 10.1006/jmbi.1995.0618 .
- Oliveberg M, Arcus VL, Fersht AR . pKA values of carboxyl groups in the native and denatured states of barnase: the pKA values of the denatured state are on average 0.4 units lower than those of model compounds . Biochemistry . 34 . 29 . 9424–9433 . July 1995 . 7626612 . 10.1021/bi00029a018 .
External links
Notes and References
- Serrano L, Kellis JT, Cann P, Matouschek A, Fersht AR . The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability . Journal of Molecular Biology . 224 . 3 . 783–804 . April 1992 . 1569557 . 10.1016/0022-2836(92)90562-X .
- Serrano L, Matouschek A, Fersht AR . The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure . Journal of Molecular Biology . 224 . 3 . 805–818 . April 1992 . 1569558 . 10.1016/0022-2836(92)90563-Y .
- Matouschek A, Serrano L, Fersht AR . The folding of an enzyme. IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure . Journal of Molecular Biology . 224 . 3 . 819–835 . April 1992 . 1569559 . 10.1016/0022-2836(92)90564-Z .
- Mossakowska DE, Nyberg K, Fersht AR . Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis . Biochemistry . 28 . 9 . 3843–3850 . May 1989 . 2665810 . 10.1021/bi00435a033 .
