The slow step of folding of Staphylococcus aureus PC1 beta-lactamase involves the collapse of a surface loop rate limited by the trans to cis isomerization of a non-proline peptide bond.
We wished to test the hypothesis that the non proline cis to trans isomerization of the peptide bond at position 167 in the S. aureus beta-lactamase PC1 exerts a significant controlling effect on the folding pathway of this enzyme. The previous data presented in support of this hypothesis could not rule out the effect of factors unrelated to non-proline cis/trans isomerization. We have used the plasmid pET9d to direct soluble overproduction of the S. aureus beta-lactamase PC1 and a site-directed mutant (Ile 167 to Pro) in Escherichia coli. Following purification the proteins were subjected to a comparative analysis of the kinetics of unfolding and refolding using the techniques of near- and far-UV circular dichroism spectroscopy and fluorescence spectroscopy in conjunction with "double-jump" experiments. Results show that the fully-unfolded I167P mutant enzyme retains 20% of molecules in a fast-refolding form and that slower-refolding molecules fold faster than the recombinant wild-type enzyme. The final stage of folding involves folding of the omega-loop into a conformation essential for enzymatic activity. In support of the original hypothesis, the folding of this omega-loop is rate limited by the isomerization of the Glu 166-Ile 167 peptide bond.
Wheeler
K A
KA
Department of Biochemistry and Genetics, Medical School, University of Newcastle Upon Tyne, United Kingdom.
Hawkins
A R
AR
Pain
R
R
Virden
R
R
D002942:Circular Dichroism; D004795:Enzyme Stability; D007536:Isomerism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D001618:beta-Lactamases