title: Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G.
abstract: The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.
authors.affiliation: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Hai-Ning
authors.initials: HN
authors.lastname: Du
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Hu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D005453:Fluorescence; D005454:Fluorescence Polarization; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D014364:Tryptophan