The cation-π interaction plays an important role in protein structure, binding, and catalytic function. 1 Despite numerous studies of the energetics of the cation-π interaction in synthetic host-guest systems, 2 its energetic value in biological systems is less well understood. 3 The measurement of cation-π energies by conventional site-directed mutagenesis is complicated by the absence of naturally occurring positively charged isosteres of neutral amino acids; nonisosteric replacements introduce perturbations that are difficult to quantify or subtract out. Consequently, we have used unnatural amino acid mutagenesis 4 with an isosteric pair of unnatural amino acids 5 to quantify the energetic contribution of an engineered cation-π interaction in the interior of staphylococcal nuclease (SNase) to overall protein stability. A hydrophobic pocket composed of two phenylalanine side chains and one tyrosine side chain is occupied in wild-type (WT) SNase by Val 74 (Scheme 1). 6 This neutral valine residue was replaced with the cationic residue S-methylmethionine using suppressor tRNA methodology (Scheme 1, B). The neutral isostere of S-methylmethionine, homoleucine, was also incorporated into position 74 for purposes of comparison (Scheme 1, A). The thermodynamic stabilities of the two mutants (which have near wild-type catalytic parameters) as well as the difference in aqueous solvation energies of the S-methylmethionine and homoleucine side chains were measured. An upper limit of 2.6 kcal mol -1 was obtained for the energy of the engineered cation-π interaction in SNase, in the absence of significant differences in packing interactions for the two mutant proteins.SNase is a 149 amino acid Ca 2+ -dependent enzyme that hydrolyzes DNA and RNA to give 3′ mono-and dinucleotides.It is a convenient system in which to examine the cation-π interaction because of the cavity formed in its hydrophobic interior by a triad of aromatic residues, Phe 34 , Phe 76 , and Tyr 27 . This cavity is partly occupied by a valine residue whose side chain extends toward the three rings; the distance from side chain to any one ring is 4-5 Å. The aromatic triad, a substructure of the five-strand -barrel which forms the major hydrophobic core of SNase, has been studied by alanine replacement mutagenesis, which showed that all three aromatic side chains interact to make an important contribution to overall protein stability. 7 To engineer a cation-π system in SNase, Val 74 was replaced with a positively charged unnatural amino acid, S-methylmethionine (S-MeMet). The trisubstituted sulfonium group of S-MeMet is sterically analogous to the neutral isopropyl functionality of valine; 8 however, to prevent -elimination of the sulfonium group, S-MeMet contains a two-carbon linker between the backbone C R and the sulfonium moiety. Modeling with X-PLOR, 9 indicates that this linker positions the sulfonium group within cation-π radius 10 of the aromatic rings. The S-MeMet amino acid was synthesized by S-methylation of N-nitroveratryloxycarbonyl-L-methionin...
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