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Probing Ligand Induced Changes in Flexibility of Glutamate Dehydrogenase Using Collisional Quenching of Fluorescence
Author(s) -
Li Chun,
Bell Ellis
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.561.6
Subject(s) - chemistry , tryptophan , quenching (fluorescence) , fluorescence , acrylamide , glutamate dehydrogenase , nad+ kinase , photochemistry , nucleotide , glutamate receptor , amino acid , biochemistry , enzyme , physics , organic chemistry , receptor , quantum mechanics , copolymer , gene , polymer
To probe the local environments of tryptophans in proteins we have developed a novel analysis of fluorescence collisional quenching using multiple emission wavelengths, combined with differently charged quenchers (acrylamide: neutral, Iodide: negative & Cesium: positive). With N Acetyl tryptophan, similar Stern‐Volmer constants are obtained at all wavelengths, as expected. With glutamate dehydrogenase, (4 tryptophans per subunit), 4 distinct families of constants are obtained with the highest occurring at long wavelengths and progressively decreasing as the wavelength band analyzed decreases showing this approach separates the effects on individual tryptophans in the protein. We have explored nucleotide effects on accessibility and charge environment of the 4 tryptophans. Results indicate that overall, nucleotide ligands NADH, NADPH and ADP significantly lower accessibility particularly in the region of subunit interfaces while GTP has less effect and in the case of W72 and W281 increases accessibility. Acrylamide quenching gives Stern‐Volmer plots concave upwards, which become linear when lifetime measurements are used. With NAD(P)H, the charged quenchers, Iodide and Cesium (but not the neutral Acrylamide) give negative slopes which appear to result from differential effects on tryptophan‐NAD(P)H resonance energy transfer. Funded by NSF Grant MCB 0448905 to EB