Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Abstract

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA-protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA-protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 +/- 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.

Effects of Molecular Crowding on the Structures, Interactions, and Functions of Nucleic Acids

“…In the study, PEG molecules, providing large negative slope values, were not appropriate for the osmotic pressure experiment probably due to the significant contribution of the excluded volume effect to the observed binding constant. The osmotic pressure studies for other DNA-binding proteins (ultrabithorax and deformed homeoproteins, tryptophan repressor protein, Hha I, and glutaminyl- and glutamyl-tRNA synthetases) also showed increased stabilities in the presence of cosolutes and decreased hydration after the association with their target sequences. − …”

A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution

“…The excitation wavelength was 295 nm and emission wavelength was 340 nm. Each point was performed separately as described before [15]. After transferring the protein from a stock solution to the cuvette, the fluorescence intensity was measured.…”

The N‐terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl‐tRNA synthetase (TyrRSapm) is a monomer in solution

“…The ligand binding was monitored by tryptophan fluorescence quenching method using Hitachi F‐7000 fluorometer in 20 mM Tris–HCl buffer (pH‐7.4) containing 16 mM MgCl 2 [31]. Constant temperature was maintained by a circulating water bath.…”