Effect of temperature on the self-assembly of the Escherichia coli ClpA molecular chaperone.
Protein quality control pathways rely upon ATP-dependent proteases, such as Escherichia coli ClpAP, to perform maintenance roles in the cytoplasm of the cell. ATP-dependent proteases remove misfolded and partially synthesized proteins. This action is particularly important in situations where an unregulated accumulation of such proteins will have a deleterious effect on the cell. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and the ATPase/protein unfoldase ClpA (84.2 kDa monomer). ClpA also uses its protein unfolding activity to remodel proteins and protein complexes; thus, in the absence of the proteolytic component, ClpA is considered a molecular chaperone. Previous reports, by others, suggested that ClpA exists in a monomer-dimer equilibrium at 4 °C. In contrast, using a combination of sedimentation velocity, sedimentation equilibrium, and dynamic light scattering, we recently reported that ClpA exists in a monomer-tetramer equilibrium at 25 °C. Here we report an investigation of the effect of temperature on the self-association of the E. coli ClpA protein unfoldase using analytical ultracentrifugation techniques. The results of sedimentation velocity and sedimentation equilibrium experiments performed at multiple loading concentrations of ClpA over a range of temperatures from 3.9 to 38.2 °C are discussed. Sedimentation velocity experiments show a decrease in weight average s(20,w) at the extremes of temperature. This result, along with extensive sedimentation equilibrium data and analysis, suggests the presence of a dimeric intermediate of ClpA that is differentially populated as a function of temperature. Further, analysis of sedimentation equilibrium data as a function of temperature led us to propose a monomer-dimer-tetramer equilibrium to describe the temperature dependence of ClpA self-assembly in the absence of nucleotide.
Veronese
P Keith
PK
Department of Chemistry, The University of Alabama at Birmingham, 1530 3RD Avenue South, Birmingham, Alabama 35294-1240, United States.
Lucius
Aaron L
AL
D019281:Dimerization; D049071:Endopeptidase Clp / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D031521:Fractionation, Field Flow / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013816:Thermodynamics; D014461:Ultracentrifugation
D029968:Escherichia coli Proteins; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp