Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.
The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.
Georgiou
G
G
Department of Chemical Engineering, University of Texas, Austin 78712.
Valax
P
P
Ostermeier
M
M
Horowitz
P M
PM
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000648:ultrastructure; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D002479:Inclusion Bodies / Q000502:physiology*; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D006146:Guanidines; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D019791:Guanidine