Unknown

Dataset Information

0

Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide.


ABSTRACT: Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. The quenching can arise from local changes near the interaction site or from binding-induced conformational changes. In cases where the titrant absorbs at or near the excitation or emission wavelengths of tryptophan, significant quenching can occur even without an interaction. This is known as the inner filter effect. This protocol describes how to use tryptophan fluorescence quenching to investigate the binding affinity of a protein for its partner/ligand and how to check and correct for the inner filter effect. As an example, we measured the binding affinity of the haem-binding protein, HusA, from Porphyromonas gingivalis for haem, and showed how we accounted for the inner filter effect.

SUBMITTER: Yammine A 

PROVIDER: S-EPMC7854220 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

altmetric image

Publications

Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide.

Yammine Anthony A   Gao Jinlong J   Kwan Ann H AH  

Bio-protocol 20190605 11


Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. The quenching can arise from local changes near the interaction site or from binding-induced conformational changes. In cases where the titrant absorb  ...[more]

Similar Datasets

| S-EPMC10543275 | biostudies-literature
| S-EPMC7289602 | biostudies-literature
| S-EPMC3936774 | biostudies-literature
| S-EPMC2758765 | biostudies-literature
| S-EPMC8654978 | biostudies-literature
| S-EPMC3938424 | biostudies-literature
| S-EPMC8654978 | biostudies-literature
| S-EPMC6694196 | biostudies-literature
| S-EPMC3439204 | biostudies-literature
| S-EPMC6396573 | biostudies-literature