ARTICLE M. Dlugosz Æ E. Blachut-Okrasin´ ska Æ E. Bojarska E. Dar _ zynkiewicz Æ J.M. Antosiewicz Effects of pH on kinetics of binding of mRNA-cap analogs by translation initiation factor eIF4E Received: 30 April 2002 / Accepted: 20 September 2002 / Published online: 31 October 2002 Ó EBSA 2002 Abstract Stopped-flow spectrofluorimetry and a theo- retical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5¢-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex. Keywords Stopped flow Æ pH dependence Æ Cap binding protein Æ Cap analogs Æ Protonation equilibria Abbreviations HEPES: N-(2-hydroxyethyl)piperazine- N¢-(2-ethanesulfonic acid); Æ MES: 2-(N-morpholi- no)ethanesulfonic acid Æ m 7 ; GMP: 7-methylguanosine 5¢-monophosphate Æ m 7 ; GDP: 7-methylguanosine 5¢-diphosphate Æ m 7 ; GTP: 7-methylguanosine 5¢-tri- phosphate Introduction It is generally recognized that electrostatic interactions constitute one of the most important determinants of binding affinity between ligands and their target re- ceptors. Ionizable groups in biomolecules represent a dominant contribution to the electrostatic potential around these molecules. Their charge state depends on pH and, as a result, ligand-receptor interactions show some degree of pH dependence. Studies of pH depen- dence are used to elucidate mechanisms of enzyme cat- alysis (Tipton and Dixon 1979; Cleland 1982; Stivers et al. 1996; Brendskag et al. 1999), as well as protein- ligand (Taylor et al. 2000) and protein-protein (Kitano et al. 1989) recognition. We here present the pH dependence of the association and dissociation rate constants of murine translation initiation factor eIF4E, associating with three analogs of the 5¢-terminal cap in mRNA: 7-methyl-guanosine 5¢-mono-, 5¢-di-, and 5¢-triphosphate (m 7 GMP, m 7 GDP, and m 7 GTP, respectively). The observations are inter- preted on the basis of a theoretical method for prediction of protonation equilibria in proteins (Antosiewicz et al. 1996; Briggs and Antosiewicz 1999). The eIF4E protein is a key participant in the regula- tion of translation in eukaryotic cells (Sonenberg and Gingras 1998). This protein specifically recognizes the m 7 GpppN cap (where p is a phosphate group and N is any nucleoside) of the 5¢-terminus of mRNA during in- itiation of translation processes. Considerable insight into the structural features of recognition of the mRNA cap by the eIF4E protein have been gained from the three-dimensional structures of its complexes with m 7 GDP: the crystal structure for murine eIF4E (Mar- cotrigiano et al. 1997) and a set of solution structures for yeast eIF4E derived from NMR data (Matsuo et al. 1997). The alkylated base is sandwiched between the side chains of two conserved tryptophans (Trp56 and Trp102 in the murine eIF4E). The interaction can be explained in terms of enhancement of p-p stacking enthalpy, because of charge transfer between the electron-deficient 7-me- thylguanine (which carries a delocalized positive charge secondary to methylation) and the electron-rich indole groups. However, not all details of interactions between polar titratable groups of the protein and the ligand have Eur Biophys J (2003) 31: 608–616 DOI 10.1007/s00249-002-0258-7 M. Dlugosz Æ E. Blachut-Okrasin´ska Æ E. Bojarska E. Dar _ zynkiewicz Æ J.M. Antosiewicz (&) Department of Biophysics, Warsaw University, Warsaw 02-089, Poland E-mail: jantosi@biogeo.uw.edu.pl Tel.: +48-22-5540727 Fax: +48-22-5540001