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Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein

¹ Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen, 9747 AG Groningen, The Netherlands
² Consiglio Nazionale delle Richerche (CNR), Instituto di Biofisica, Area della Ricerca di Pisa, 56010, Pisa, Italy

Reprint requests to: Jaap Broos, Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; e-mail: J.Broos{at}chem.rug.nl; fax: 31-50-3634165.

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII^mtl). EII^mtl overexpression was under control of the P_R promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the P_R promoter. Together with the presence of the repressor gene on the EII^mtl plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EII^mtl samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs.

Keywords: Tryptophan analog; biosynthetic incorporation; alloprotein; phosphorescence spectroscopy; membrane protein

Abbreviations: EII^mtl, the mannitol-specific transporting and phosphorylating enzyme from Escherichia coli • Mtl, mannitol • Trp, tryptophan • 1-MTrp, 1-methyltryptophan • 4-FTrp, 4-fluorotryptophan • 5-FTrp, 5-fluorotryptophan • 5-OHTrp, 5-hydroxytryptophan • 7-ATrp, 7-azatryptophan • 4-Findole, 4-fluoroindole • 5-Findole, 5-fluoroindole • 5-OHindole, 5-hydroxyindole • C₁₀E₅, decylpenta (ethylene glycol) • NATA, N-acetyl-L-tryptophanamide • PG, 1,2 propylene glycol • BW, bandwidth in nm of the 0,0 vibronic band at two-thirds peak height • _0,0, peak wavelength of the 0,0 vibrational band • _ex, excitation wavelength • _p, phosphorescence quantum yield

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