Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein.
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 lambdaP(R) promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the lambdaP(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.
Broos
Jaap
J
Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen, 9747 AG Groningen, The Netherlands. J.Broos@chem.rug.nl
Gabellieri
Edi
E
Biemans-Oldehinkel
Esther
E
Strambini
Giovanni B
GB
D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007211:Indoles / Q000737:chemistry*; D008163:Luminescent Measurements; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
C023422:7-azaindole dimer; D007211:Indoles; D014364:Tryptophan