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Stability and unfolding pathway of E. coli periplasmic proteins monitored by F-19 NMR and fluorescence spectroscopy

The Leucine-isoleucine-valine binding protein (LIV) and the Leucine-specific binding protein (LS) are soluble receptors in the hydrophobic amino acid transport systems of Escherichia coli. They have been used as structural models of several human neuronal receptors. We investigated the stability and unfolding model for those two proteins using F-19 NMR and fluorescence spectroscopies. The tryptophan residues were used to monitor urea induced unfolding in wild type and Trp to Phe mutants. Reversible unfolding was found for the wild type and all mutant proteins. Thermodynamic parameters were obtained for both periplasmic proteins. Single sigmoidal transitions were observed for the proteins without ligands, since the proteins underwent a distinct two-state unfolding. Ligand binding imparts stabilization to the protein, and the unfolding curves showed a three-state mechanism where one lobe unfolds before the other. LIV appeared to be more stable than LS and L-Ile afforded the most stable complex. The disulfide bond is important for stabilization of the proteins. A decrease in Gibbs free energy was observed when that bond was broken in unliganded LIV and LS by the addition of DTT. Based on urea unfolding of Trp mutants, an unfolding model was proposed. The N-lobe was observed to unfold first, while the C-lobe was more stable to urea denaturation. F-19 NMR studies showed that the protein twists to the open form and then each lobe unfolds. The ligand appears to remain bound to the N-lobe near W-18 after cleft opening

Escherichia coli; Leucine-isoleucine-valine binding protein; Leucine-specific binding protein; mutant protein; urea-induced unfolding; F-19 NMR; fluorescence spectroscopy

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