Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA.
The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.
Talavera
Miguel A
MA
Yale University, Department of Molecular Biophysics & Biochemistry, 260 Whitney Avenue, New Haven, Connecticut 06520, USA.
De La Cruz
Enrique M
EM
D000227:Adenine Nucleotides / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D020365:RNA Helicases / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D000227:Adenine Nucleotides; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; C065908:dbpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases