title: Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetradecamer with an axial pore.
abstract: 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.
authors.affiliation: Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Flanagan
authors.forename: J S
authors.initials: JS
authors.lastname: Wall
authors.forename: M S
authors.initials: MS
authors.lastname: Capel
authors.forename: D K
authors.initials: DK
authors.lastname: Schneider
authors.forename: J
authors.initials: J
authors.lastname: Shanklin
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D017348:Microscopy, Electron, Scanning Transmission; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases