Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy.
The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.
Laboratory of Biophysical Chemistry and Groningen Biomolecular Science and Biotechnology Institute-GBB, University of Groningen, 9747 AG Groningen, The Netherlands.
Dejan M
DM
Petrović
Kees
K
Leenhouts
Maarten L
ML
van Roosmalen
Fenneke
F
Kleinjan
Jaap
J
Broos
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D013294:Lactococcus lactis / Q000201:enzymology*; D008024:Ligands; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D017186:Titrimetry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism
D002241:Carbohydrates; D008024:Ligands; D014364:Tryptophan; C092497:AcmA protein, Lactococcus lactis; D009113:Muramidase