In vitro site-specific incorporation of fluorescent probes into β-galactosidase

Journal of the American Chemical Society

Volumn 119, Issue 1, 1997, Pages 6-11

  Steward, Lance E ^a   Collins, Cynthia S ^a   Gilmore, Marcella A ^a   Carlson, Justin E ^b   Ross, J B Alexander ^b   Chamberlin, A Richard ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b MOUNT SINAI SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BETA GALACTOSIDASE;

AMINO ACID COMPOSITION; ARTICLE; ENZYME STRUCTURE; FLUORESCENCE; SITE DIRECTED MUTAGENESIS; TECHNIQUE;

EID: 0031042616     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja963023f     Document Type: Article

References (50)

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8. Difference spectroscopy is one powerful technique that has recently been developed for deciphering intrinsic Tip fluorescence in multi-Trp proteins. See: Hasselbacher, C. A.; Rusinova, E.; Waxman, E.; Rusinova, R.; Kohanski, R. A.; Lam, W.; Guha, A.; Du, J.; Lin, T. C.; Polikarpov, I.; Boys, C. W. G.; Nemerson, Y.; Konigsberg, W. H.; Ross, J. B. A. Biophys. J. 1995, 69, 20-29.
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12. (a) This has been accomplished with Trp deficient bacterial auxotrophs. See Ross, J. B. A.; Szabo, A. G.; Hogue, C. W. V. Methods Enzymol., in press, and references cited therein. (b) The site-specific incorporation of amino acid 3 into T4 lysozyme by suppression techniques has also previously been reported: Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2910-2914.
13. (a) This has been accomplished with Trp deficient bacterial auxotrophs. See Ross, J. B. A.; Szabo, A. G.; Hogue, C. W. V. Methods Enzymol., in press, and references cited therein. (b) The site-specific incorporation of amino acid 3 into T4 lysozyme by suppression techniques has also previously been reported: Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2910-2914.
14. Examination of the X-ray crystal structure reveals that the majority of the 20 Lys residues per monomer are on the protein surface: Jacobson, R. H.; Zhang, X.-J.; DuBose, R. F.; Matthews, B. W. Nature 1994, 369, 761-766.
15. It should be mentioned that other amino acids modified with fluorescent reagents have also been incorporated biosynthetically, but either in systems that do not allow site-specificity or in systems that are more limited. See: (a) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell 1994, 78, 461-471. (b) Crowley, K. S.; Reinhart, G. D.; Johnson, A. E. Cell 1993, 73, 1101-1115. (c) Picking, W. D.; Picking, W. L., Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 2368-2375. (d) Picking, W. D.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 12565-12570 and references cited therein.
16. It should be mentioned that other amino acids modified with fluorescent reagents have also been incorporated biosynthetically, but either in systems that do not allow site-specificity or in systems that are more limited. See: (a) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell 1994, 78, 461-471. (b) Crowley, K. S.; Reinhart, G. D.; Johnson, A. E. Cell 1993, 73, 1101-1115. (c) Picking, W. D.; Picking, W. L., Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 2368-2375. (d) Picking, W. D.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 12565-12570 and references cited therein.
17. It should be mentioned that other amino acids modified with fluorescent reagents have also been incorporated biosynthetically, but either in systems that do not allow site-specificity or in systems that are more limited. See: (a) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell 1994, 78, 461-471. (b) Crowley, K. S.; Reinhart, G. D.; Johnson, A. E. Cell 1993, 73, 1101-1115. (c) Picking, W. D.; Picking, W. L., Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 2368-2375. (d) Picking, W. D.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 12565-12570 and references cited therein.
18. It should be mentioned that other amino acids modified with fluorescent reagents have also been incorporated biosynthetically, but either in systems that do not allow site-specificity or in systems that are more limited. See: (a) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell 1994, 78, 461-471. (b) Crowley, K. S.; Reinhart, G. D.; Johnson, A. E. Cell 1993, 73, 1101-1115. (c) Picking, W. D.; Picking, W. L., Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 2368-2375. (d) Picking, W. D.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 12565-12570 and references cited therein.
19. The active enzyme is a homotetramer comprised of 116 kDa (1023 amino acid) monomers. See: (a) Fowler, A. V.; Zabin, I. J. Biol. Chem. 1978, 253, 5521-5525. (b) Kalnins, A.; Otto, R.; Ruther, U.; Muller-Hill, B. Embo J. 1983, 2, 593-597.
20. The active enzyme is a homotetramer comprised of 116 kDa (1023 amino acid) monomers. See: (a) Fowler, A. V.; Zabin, I. J. Biol. Chem. 1978, 253, 5521-5525. (b) Kalnins, A.; Otto, R.; Ruther, U.; Muller-Hill, B. Embo J. 1983, 2, 593-597.
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24. Studies of lacZ gene fusions have shown that up to 26 amino acids can be removed from the amino terminus and can be substituted by other amino acid sequences with little or no effect on the specific activity: Fowler, A. V.; Zabin, I. J. Biol. Chem. 1983, 258, 14354-14358. This allows noncoded amino acids to be screened efficiently without fear that decreased activity is due to structural changes. Additionally, since termination competes with suppression to give only a hexapeptide, there is no possibility of the termination product having enzymatic activity.
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30. A detailed comparision is underway.
31. This difference could be due to different proteins or different codon context, as well as the use of the temperature-sensitive RF strain.
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38. (d) As an additional control for hydrophobic binding (or adsorption) of ∈-dnsLys, wild-type β-galactosidase produced in a control S-30 lysate reaction was incubated in the presence of ∈-dnsLys. The wt enzyme was then purified as described and the fluorescence spectrum recorded. It is shown, in figure 1, that no significant amount of ∈-dnsLys is present in the purified wt β-galactosidase, further evidence that the fluorescence emission of dnsLys-β-galactosidase is due to ∈-dnsLys that is incorporated into the peptide chain.
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40. See, for example: (a) Gottikh, B. P.; Krayevsky, A.; Tarussova, N. B.; Purygin, P. P.; Tsilevich, T. L. Tetrahedron 1970, 26, 4419-4433. (b) Tarusova, N. B.; Mazurova, V. V.; Kraevskii, A. A.; Gottikh, B. P. Bull. Acad. Sci. USSR, Div. Chem. Sci. 1971, 1630-1633 and preceeding paper in the series.
41. See, for example: (a) Gottikh, B. P.; Krayevsky, A.; Tarussova, N. B.; Purygin, P. P.; Tsilevich, T. L. Tetrahedron 1970, 26, 4419-4433. (b) Tarusova, N. B.; Mazurova, V. V.; Kraevskii, A. A.; Gottikh, B. P. Bull. Acad. Sci. USSR, Div. Chem. Sci. 1971, 1630-1633 and preceeding paper in the series.
42. The two peaks correspond to the 2′- and 3′-isomers.
43. Amit, B.; Sehavi, V.; Patchornik, A. J. Org. Chem. 1974, 39, 192-196.
44. Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J. Methods Enzymol. 1991, 202, 301-336.
45. Steers Jr., E.; Cuatrecasas, P. Methods Enzymol. 1974, 34, 350-358.
46. Binding capacity reported by Sigma: 2-8 mg of β-galactosidase/ mL of resin.
47. Background readings normally returned to baseline after washing with 2 mL of buffer; however, each column was washed with a minimum of 5 mL.
48. The assay can be run on the 800 μL quenched reaction; however, in order for the assay to be accurate, the quenched reaction should be diluted such that the formation of yellow color resulting from hydrolysis of ONPG should take at least 10 min. Thus high yielding reactions must be diluted several-fold. The amount of dilution is determined experimentally but is generally within the 2-100-fold range (400-8 μL, respectively, of the quenched reaction).
49. 0C. Pure β-galactosidase has an activity of ca. 300 000 units/mg. See ref 14.
50. 0C for convenience.