ProteinProtein Interaction Using Tryptophan Analogues: Novel Spectroscopic Probes for ToxinElongation Factor-2 Interactions

Publisher Website
Google Scholar
Abstract
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 Pseudomonasaeruginosa [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 Escherichiacoli, 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 kcat 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) Biochemistry35, 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.