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Monitoring the unfolding pathway of the L-leucine binding proteins of E. coli by F-19 NMR and fluorescence spectroscopy

Two L-leucine binding proteins of E. coli with overlapping specificities for branched chain amino acids are found in the periplasmic space. There they serve as the first receptors for transport across the membrane. Structurally they have two domains that are open when ligand is not present. Binding of ligand induces a conformational change to a closed form, which is recognized by the membrane complex. There has been a growing interest in these proteins since they have been used as structural models for several neuronal proteins including the Group I metabotrophic glutamate receptors and the N-methyl-D-aspartate receptor. Our laboratory has found that the 'so called' branched amino acid receptors are much more promiscuous in their binding capacities. The L-leucine specific protein has been shown to bind L-phenylalanine and fluoro derivatives of phenylalanine, which are similar in structure to the agonists and antagonists of the glutamate receptors. There may be more similarities in these two families of receptors than was first postulated. We have investigated the unfolding pathways of the two leucine binding proteins of E. coli in urea by intrinsic fluorescence. Thermodynamic stabilities have been determined using a three state model that interprets the unfolding curve. Using site directed mutagenesis of each tryptophan residue we have been able to determine the pathway of unfolding of these two domain proteins. In addition, 5-fluorotryptophan labeled proteins have allowed F-19 NMR spectroscopy to further corroborate our unfolding pathways.

L-leucine binding proteins; Escherichia coli; urea-induced unfolding; 5-fluorotryptophan labeling; fluorine (F-19) NMR; fluorescence spectroscopy

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