engleski

Mutagenesis, NMR and protein unfolding studies on the leucine receptor of E. coli

Periplasmic binding proteins serve as initial receptors of active transport for a wide variety of substrates. These proteins can be used as models for many receptor-ligand interactions such as those observed in the steroid receptors. Two proteins which bind hydrophobic-branched amino acids, the leucine/isoleucine/valine-binding protein (LIV) and the leucine-specific binding proteins (LS) share the same overall structure but have different specificities. One of the most obvious differences between these two proteins is position 18 where LS has a Trp residue and LIV has Tyr. To address this critical variance and further understand the method of binding differentiation, mutants (LSW18Y, LSW18F, LSW320F, LSW320+336F, LSW336F, LSW278F) were made for fluorescence, UV, titration calorimetry and fluorine NMR studies. Our work has demonstrated that LS and LIVY18W have a much wider range of substrates including phenylalanine and fluorinated analogues of phenylalanine. NMR studies have shown that Trp18 is involved in binding of the substrates. In addition, mutants LSW330F and LS336F show greater motional properties than the wild type. We also have observed a large hinge-bending motion that shows two dissimilar conformations when leucine and phenylalanine bind to the cleft. The hydrophobic receptors may be more flexible than other periplasmic proteins and may show the same promiscuous binding properties as the steroid receptors. Denaturation studies show the ligand bound form to be more stable. Although phenylalanine binds to the LS protein it does not seem to bind in the same manner as leucine.

leucine/isoleucine/valine-binding protein; leucine specific binding protein; conformational analysis; NMR-spectroscopy; auxotropic mutants

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