Protein-protein interaction using tryptophan analogues: novel spectroscopic probes for toxin-elongation factor-2 interactions.
Previously, we characterized the role of the three naturally occurring Trp residues (W-417, -466, and -558) in the catalytic mechanism of the toxin-enzyme produced by Pseudomonas aeruginosa [Beattie and Merrill (1999) J. Biol. Chem. 274, 15646-15654]. However, the use of intrinsic Trp fluorescence to study toxin-eEF-2 interaction is inherently limited since the spectral properties of the various Trp residues in both proteins cannot easily be distinguished. To facilitate the study of the protein-protein interaction by Trp fluorescence spectroscopy, the Trp residues in the catalytic domain of exotoxin A were replaced with the amino acid analogues 4-fluorotryptophan, 5-fluorotryptophan, 5-hydroxytryptophan, and 7-azatryptophan. The incorporation of analogues was achieved by using a tightly regulated promoter, pBAD, and expressing the protein in a Trp auxotrophic strain of Escherichia coli, BL21, in a minimal medium containing the appropriate tryptophan analogue. Quantitative spectral analysis of the analogue-containing proteins using the Decompose program indicated that we had achieved 87-100% incorporation efficiency depending on the Trp analogue being used. Electrospray mass spectrometry analysis verified that we had achieved nearly total replacement of the L-tryptophan residues within the catalytic domain of exotoxin A with the tryptophan analogues 5-fluorotryptophan and 4-fluorotryptophan. The analogue-substituted proteins showed a variation in their catalytic activities with k(cat) values ranging from 6-fold (4-fluorotryptophan) to 260-fold (5-hydroxytryptophan) lower than the natural enzyme, which was in agreement with previous data using site-directed mutagenesis [Beattie et al. (1996) Biochemistry 35, 15134-15142]. However, the analogue-incorporated enzymes did not show any significant change in their ability to bind NAD(+) as substrate, as determined from a fluorescence-binding assay. The spectral properties of the various analogue-incorporated proteins were evaluated and compared with those of the native protein. Furthermore, selective excitation of the 5-hydroxytryptophan-incorporated toxin was exploited to study its interaction with the elongation factor-2 substrate by fluorescence resonance energy transfer to an acceptor chromophore located on the elongation factor-2 protein. The binding between the toxin-enzyme and elongation factor-2 was shown to be independent of the NAD(+) substrate (983 +/- 63 nM) and showed a small dependence upon the ionic strength of the solution.
Mohammadi
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F
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario N1G 2W1, Canada.
Prentice
G A
GA
Merrill
A R
AR
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000737:chemistry* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D014364:Tryptophan; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D009243:NAD; D020652:Peptide Elongation Factor 2; D011506:Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A