Unfolded lactamase

The removal of the signal sequence which directs the transport of secreted proteins is a critical step in protein export. Extensive studies by Randall, Hardy and coworkers have demonstrated that the leader sequence prevents the proper folding of the mature polypeptide within the cytoplasm (55,54). For the E.coli 6-lactamase, the presence of the leader sequence decelerates the folding kinetics but does not prevent the formation of the enzymatically active conformation (55). Bowden and Georgiou (56) showed that the mode of translocation of 6-lactamase across the cytoplasmic membrane of Escherichia coli exerts a profound effect on the folding of the mature protein following secretion, presumably by affecting the unfolded state in the periplasmic space. 

Folding Equilibrium Studies. E. coli RTEM p-lactamase is a monomeric protein. Its amino acid sequence has been determined (79). It has one disulfide bond between the residues Cys S and Cys. The presence of four tyrosines and four tryptophans allows the use of spectroscopic method for the conformational characterization of the enzyme. In this study, the effect of denaturants on the unfolding of p-lactamase was determined from activity measurements, difference spectroscopy and fluorescence intensity measurements. 

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

A simple linear regression was used to fit the data obtained by fluorescence measurements (Figure 4). AGy(KH2PO4), the energy of unfolding of p-lactamase in... 

Renaturation. The effects of i) reduction of the disulfide bond of p-lactamase, ii) renaturation buffer pH, iii) concentration of protein, iv) GuHCl in the denaturing solution and finally v) sucrose concentration on the reversibility of the unfolding transition were investigated. 

The denaturation equilibrium experiments described in this paper show that p-lactamase becomes unfolded in the presence of more than 1.25 M guanidine-HCl. The noncoincidence of the difference spectra and fluorescence emission at guanidine-HCl concentrations between 0.3 and 0.75 M suggests the presence of an intermediate which is populated at equilibrium. 

Figure 7. Percent of enzymatic activity recovered upon renaturation of 10 mg/ml of p-lactamase unfolded in different concentrations of GuHCl. The unfolding buffer contained 5 mM DTT in 50 mM potassium phosphate, pH 7.0 in addition to the denaturant. The protein was refolded by didysis against 50mM potassium phosphate, pH 6.0 at 23 C. The final GuHCl concentration was 0.02 M for all experiments.

Figure 8. Effect of sucrose concentration on the renaturation of p-lactamase. Samples with different concentrations of protein were unfolded in 2.0 M GuHCl, 5 niM DTT and renatured by dialysis in potassium phosphate buffer, pH 6.0 (0.02 M final GuHCl concentration) with 0 M ( ), 0.15 M ( ) and (o) 0.3 M sucrose...

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