Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

Dejan M. Petrovic, Kees Leenhouts, Maarten L. van Roosmalen, Fenneke KleinJan, Jaap Broos*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

12 Citaten (Scopus)
53 Downloads (Pure)


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. (C) 2012 Elsevier Inc. All rights reserved.

Originele taal-2English
Pagina's (van-tot)111-118
Aantal pagina's8
TijdschriftAnalytical Biochemistry
Nummer van het tijdschrift2
StatusPublished - 15-sep-2012

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