Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E.
Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.
Niedzwiecka
Anna
A
Department of Biophysics, Institute of Experimental Physics, Warsaw University, 02-089 Warszawa, Poland.
Darzynkiewicz
Edward
E
Stolarski
Ryszard
R
D000818:Animals; D002412:Cations / Q000737:chemistry; D019277:Entropy; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry* / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006358:Hot Temperature; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012314:RNA Cap Analogs / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D002412:Cations; D039561:Eukaryotic Initiation Factor-4E; D021122:Protein Subunits; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D006151:Guanosine; C016578:7-methylguanosine; D014364:Tryptophan