Loop-directed mutagenesis converts amicyanin from Thiobacillus versutus into a novel blue copper protein

Journal of the American Chemical Society

Volumn 118, Issue 31, 1996, Pages 7406-7407

  Dennison, Christopher ^a   Vijgenboom, Erik ^a   Hagen, Wilfred R ^b   Canters, Gerard W ^a  

^a LEIDEN UNIVERSITY   (Netherlands)

^b WAGENINGEN UNIVERSITY   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

AMICYANIN;

AMINO ACID SEQUENCE; ARTICLE; MUTANT; PROTEIN STRUCTURE; SITE DIRECTED MUTAGENESIS; SPECTROPHOTOMETRY;

EID: 0029759527     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja953256r     Document Type: Article

References (24)

1. Sykes, A. G. Adv. Inorg. Chem. 1991, 36, 377-408.
2. Solomon, E. I.; Baldwin, M. J.; Lowery, M. D. Chem. Rev. 1992, 92, 521-542.
3. Adman, E. T. Adv. Protein Chem. 1991, 42, 144-197.
4. Chapman, S. K. In Perspectives on Bioinorganic Chemistry; Hay, R. W., Dilworth, J. R., Nolan, K. B., Eds.; Jai Press Ltd.: London, 1991; pp 95-140.
5. Dennison, C.; Kohzuma, T.; McFarlane, W.; Suzuki, S.; Sykes, A. G. Inorg. Chem. 1994, 33, 3299-3305.
6. Canters, G. W.; Gilardi, G. FEBS Lett. 1993, 325, 39-48.
7. The amino acid sequence from His96 to Met99 in wild-type amicyanin was replaced with the sequence His-Gln-Gly-Ala-Gly-Met as found in poplar plastocyanin (see Table 1) using a modified version of the unique-site elimination mutagenesis protocol (Deng, W. P.; Nickoloff, J. A. Anal. Biochem. 1992, 260, 81-88). The mutation was verified by sequencing pCD5.
8. Expression, isolation, and purification of CD5 were carried out as for the wild-type protein (Kalverda, A. P.; Wymenga, S. S.; Lommen, A.; Van de Ven, F. J. M.; Hilbers, C. W.; Canters, G. W. J. Mol. Biol. 1994, 240, 358-371) except that an additional purification step, involving a mono-Q column (FPLC) at pH 7.5, was introduced.
9. 3- at pH 7.0 in 100 mM phosphate buffer (for the titration procedure, see: van de Kamp, M.; Silvestrini, M. C.; Brunori, M.; van Beeumen, J.; Hali, F. C.; Canters, G. W. Eur. J. Biochem. 1990, 194, 109-118).
10. The EPR parameters for CD5 amicyanin were obtained from simulations.
11. Han, J.; Loehr, T. M.; Lu, Y.; Valentine, J. S.; Averill, B. A.; Sanders-Loehr, J. J. Am. Chem. Soc. 1993, 115, 4256-4263.
12. Lu, Y.; LaCroix, L. B.; Lowery, M. D.; Solomon, E. I.; Bender, C. J.; Peisach, J.; Roe, J. A.; Gralla, E. B.; Valentine, J. S. J. Am. Chem. Soc. 1993, 115, 5907-5918.
13. Romero, A.; Hoitink, C. W. G.; Nar, H.; Huber, R.; Messerschmidt, A.; Canters, G. W. J. Mol. Biol. 1993, 229, 1007-1021.
14. Karlsson, B. G.; Nordling, M.; Pascher, T.; Tsai, L. C.; Sjölin, L.; Lundberg, L. G. Protein Eng. 1991, 4, 343-349.
15. Kalverda, A. P.; Salgado, J.; Dennison, C.; Canters, G. W. Biochemistry 1996, 35, 3085-3092.
16. Ai, J.; Dennison, C.; Canters, G. W.; Sanders-Loehr, J. Unpublished results. See: Andrew, C. R.; Yeom, H.; Valentine, J. S.; Karlsson, B. G.; Bonander, N.; van Pouderoyen, G,; Canters, G. W.; Loehr, T.; Sanders-Loehr, J. J. Am. Chem. Soc. 1994, 116, 11489-11498.
17. Ai, J.; Dennison, C.; Canters, G. W.; Sanders-Loehr, J. Unpublished results. See: Andrew, C. R.; Yeom, H.; Valentine, J. S.; Karlsson, B. G.; Bonander, N.; van Pouderoyen, G,; Canters, G. W.; Loehr, T.; Sanders-Loehr, J. J. Am. Chem. Soc. 1994, 116, 11489-11498.
18. Lommen, A.; Canters, G. W. J. Biol. Chem. 1990, 265, 2768-2774.
19. The reduction potential was measured as a function of pH using cyclic voltammetry with the measurements carried out using the apparatus and procedures as described in: Hagen, W. R, Eur. J. Biochem. 1989, 182, 523-530.
20. The peak separation in the cyclic voltammograms of the CD5 mutant of amicyanin were approximately 100 mV compared to the more ideal value of 60 mV in the case of the wild-type protein. At pH values below 7.0, the electrochemical response of the CD5 mutant at a bare glassy carbon electrode deteriorated.
21. 3-. Values of 310 mV in 100 mM phosphate buffer at pH 7.0 (I = 0.223 M) and of 357 mV in 100 mM acetate buffer at pH 5.3 (I = 0.223 M, NaCl) have been determined (see ref 9).
22. ∈1H assigned on the basis of its greater pH dependence. Due to a pH-induced conformational change at the active site, the resonances of the ring protons of His54 of CD5 also titrate, but in this case the difference between the resonance frequency in the deprotonated and protonated forms is much less. This is a very similar situation to that found previously for the wild-type protein (see ref 17 and Lommen, A.; Wijmenga, S.; Hilbers, C. W.; Canters, G. W. Eur. J. Biochem. 1991, 201, 695-702.
23. Suzuki, S.; Sakurai, T.; Shidara, S.; Iwasaki, H. Inorg. Chem. 1989, 28, 802-804.
24. Dennison, C.; Kohzuma, T.; McFarlane, W.; Suzuki, S.; Sykes, A. G. J. Chem. Soc. Dalton Trans. 1994, 437-443.