The pro-sequence domain of streptopain directs the folding of the mature enzyme.
The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain.
Anderson
Elizabeth T
ET
Department of Protein Chemistry, Wyeth Research, 401 N. Middletown Road, Bldg. 205/228, Pearl River, NY 10965, USA.
Winter
Laurie A
LA
Fernsten
Phil
P
Olmsted
Stephen B
SB
Matsuka
Yury V
YV
D001665:Binding Sites; D015153:Blotting, Western; D002845:Chromatography; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature; D013997:Time Factors
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C042477:streptopain