Scanning Transmission Electron Microscopy and Small-Angle Scattering Provide Evidence that Native Escherichia coli ClpP is a Tetradecamer with an Axial Pore

The Escherichia coli ATP-dependent caseinolytic protease (Clp) is composed of two distinct subunits; protease, ClpP, and ATPase, ClpA. Active ClpP has been overexpressed to approximately 50% of soluble protein in E. coli, and purified to homogeneity. Direct mass determination of individual particles...

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Bibliographic Details
Published in:Biochemistry (Easton) 1995-08, Vol.34 (34), p.10910-10917
Main Authors: Flanagan, John M, Wall, Joseph S, Capel, Malcolm S, Schneider, Dieter K, Shanklin, John
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - chemistry
Adenosine Triphosphatases - genetics
Adenosine Triphosphatases - isolation & purification
Adenosine Triphosphatases - ultrastructure
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Base Sequence
Endopeptidase Clp
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins
Microscopy, Electron, Scanning Transmission
Models, Molecular
Molecular Sequence Data
Molecular Weight
Protein Conformation
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - ultrastructure
Scattering, Radiation
Serine Endopeptidases - chemistry
Serine Endopeptidases - genetics
Serine Endopeptidases - isolation & purification
Serine Endopeptidases - ultrastructure
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Summary:The Escherichia coli ATP-dependent caseinolytic protease (Clp) is composed of two distinct subunits; protease, ClpP, and ATPase, ClpA. Active ClpP has been overexpressed to approximately 50% of soluble protein in E. coli, and purified to homogeneity. Direct mass determination of individual particles using scanning transmission electron microscopy (STEM) yields a mean native molecular mass of 305 +/- 9 kDa for the ClpP oligomer, suggesting that it has a tetradecameric structure. Small-angle X-ray scattering (SAXS) curves were determined for ClpP in solution at concentrations of 1-10 mg/mL. A combination of STEM and SAXS data was used to derive a model for ClpP, comprising a cylindrical oligomer about 100 A in diameter and about 75 A in height with an axial pore about 32-36 A in diameter. The volume of the pore is estimated to be approximately 70,000 A3, similar in size to those found in chaperone proteins, and is large enough to accommodate unfolded polypeptide chains, although most globular folded proteins would be excluded.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00034a025