The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.
Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) undergoes an autocatalytic process to generate 44.9 and 21.7 kDa subunits; however, a mutant protein (T399A) loses completely the processing ability and mainly exists as a precursor. For a comprehensive understanding of their structural features, the biophysical properties of these two proteins were investigated by circular dichroism and fluorescence spectroscopy. Tryptophan fluorescence and circular dichroism spectra were nearly identical for BlGGT and T399A, but unfolding analyses revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and the unprocessed T399A displayed T(m) values of 61.4°C and 68.1°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Fluorescence quenching analysis showed that T399A had a dynamic quenching constant similar to that of the wild-type enzyme. BlGGT started to unfold beyond ∼2.14 M GdnHCl and reached an unfolded intermediate, [GdnHCl](0.5, N - U), at 2.85 M, corresponding to free energy change [Formula: see text] of 12.34 kcal mol( - 1), whereas the midpoint of the denaturation curve for T399A was approximately 3.94 M, corresponding to a [Formula: see text] of 4.45 kcal mol( - 1). Taken together, it can be concluded that the structural stability of BlGGT is superior to that of T399A.
A CP1201 RIR1 intein is found in the ribonucleotide reductase alpha subunit (RNR α subunit) protein of lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078. This intein can be over-expressed and spliced in Escherichia coli NovaBlue cells. Mutations of C539, the N-terminal residue of the C-extein in the CP1201 RIR1 protein, led to the changes of pattern and level of protein-splicing activities. A G392S variant was found to be a temperature-sensitive protein with complete splicing activity at 17 and 28°C but not at 37°C or higher. We also found that the cleavage at the CP1201 RIR1 intein C-terminus of the double mutant G392S/C539G was blocked, but other cleavage activities could be efficiently performed at 17°C. G392S/C539G variant possessed the properties of low-temperature-induced cleavage at the intein N-terminus.
To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the alpha-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.
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