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 comp osed of two distinct subunits; protease, ClpP, and ATPase, ClpA. Activ e 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 Clp P oligomer, suggesting that it has a tetradecameric structure. Small-a ngle X-ray scattering (SAXS) curves were determined for ClpP in soluti on at concentrations of 1-10 mg/mL. A combination of STEM and SAXS dat a was used to derive a model for ClpP, comprising a cylindrical oligom er about 100 Angstrom in diameter and about 75 Angstrom in height with an axial pore about 32-36 A in diameter. The volume of the pore is es timated to be similar to 70 000 Angstrom(3) similar in size to those f ound in chaperone proteins, and is large enough to accommodate unfolde d polypeptide chains, although most globular folded proteins would be excluded.