Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy.
Anal Biochem
; 428(2): 111-8, 2012 Sep 15.
Article
em En
| MEDLINE
| ID: mdl-22713342
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Triptofano
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Muramidase
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Lactococcus lactis
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article