Photophysical properties of popular fluorescent adenosine nucleotide analogs used in enzyme mechanism probing.
Fluorescent nucleotide analogs are widely used in mechanistic studies of nucleotide binding and utilizing proteins. We describe here an overview of the photophysical parameters of the most popular nucleotide analogs that have a fluorescent N-methylanthraniloyl-group attached at various positions of the nucleotide. Steady state absorption and fluorescence spectra of free chromophores depend on the type of modification (ribose, base or phosphate moiety) and the addition of proteins suggests that the labeled nucleotides also vary in sensitivity depending upon their local protein environment. Fluorescence lifetime measurements imply two to three lifetimes for each nucleotide with complex changes in dependence on solvent but more importantly also on the protein. The measured quantum yields quantify the increase in fluorescence for (C8)-MABA-ADP, MANT-ATP and (Pgamma)-MABA-ATP as 153%, 93% and 14% when bound to DnaK, ClpB and Trap1, respectively, compared to free in buffer solution.
Leskovar
Adriane
A
Max-Planck-Institute for Medical Research, Department of Biomolecular Mechanisms, Jahnstrasse 29, D-69120 Heidelberg, Germany.
Reinstein
Jochen
J
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011817:Rabbits; D016963:Thermus thermophilus / Q000201:enzymology*; D014364:Tryptophan; D062367:ortho-Aminobenzoates / Q000378:metabolism
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C105264:N(8)-(4-((N'-methylanthraniloyl)amino)butyl)-8-aminoadenosine 5'-diphosphate; C092306:TRAP1 protein, human; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli