Analysis Of Sedimentation Equilibrium Data Research Articles

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.

Self-association of CPV3, an avian thymic parvalbumin

The avian parvalbumin called CPV3 readily forms disulfide-linked oligomers. Sedimentation data presented herein reveal that CPV3 also undergoes noncovalent self-association. Interestingly, the noncovalent interaction is promoted by either Ca 2+ or Mg 2+, whereas covalent complex formation displays an absolute requirement for the Ca 2+-bound protein. Apo-CPV3 exhibits an apparent sedimentation coefficient of 2.08 S at 20°C, in 0.15 M NaCl, 0.025 M HEPES-NaOH, pH 7.4. This value increases to 2.85 S or 3.16 S with addition of 1.0 mM Ca 2+ or 5.0 mM Mg 2+, respectively. Least-squares analysis of sedimentation equilibrium data suggests that 100 μM apo-CPV3 is primarily a mixture of monomeric and dimeric forms. With the addition of Ca 2+, the equilibrium becomes exclusively monomer-trimer, with negligible amounts of dimer. A comparable distribution is observed in the presence of Mg 2+.

Dimerization of Native and C-Terminally Proteolyzed p56 lck Tyrosine Kinase

Recombinant p56 lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56 lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M r ≍ 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56 lck gave M r′s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 μM. Purified intact p56 lck incorporated 2 mol of [ 32P] P i per mole of enzyme. Purified processed p56 lck incorporated only 1 mol of [ 32P] P i per mole of enzyme. The loss of 1 mol of [ 32P] P i per mole of enzyme after thrombin deletion of the C-terminus demonstrates that p56 lck undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory phosphorylation site, but which now must also be considered as an autophosphorylation site.

Average quantities accessible to the analysis of sedimentation equilibrium data for self-association systems

This paper shows that analysis of sedimentation equilibrium data, searching for average molecular weights, gives quantities which are dependent on the total protein concentration of the samples, while knowledge of the molecular weight of the monomeric subunits allows a more meaningful search for the concentrations of the individual polymeric components of the system. From these the various average molecular weights can be construed, and the various dissociation equilibrium constants evaluated. Also, in this paper considerations are proposed on the meaning of nonideality terms in associating systems and possible ways for estimating them. As an example the proposed procedures have been applied to measurements of sedimentation equilibrium in carbonmonoxyhemoglobin.

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering, sedimentation, and circular dichroism studies on the holo- and apoproteins

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 Å) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 Å 3), surface (11,840 ± 120 vs 13,470 ± 140 Å), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H 2O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.

The molecular weight and subunit structure of human erythrocyte 6-phosphogluconate dehydrogenase.

6-Phosphogluconate dehydrogenase from human erythrocytes exists predominantly as a species of 104000 molecular weight under various conditions of pH and ionic strength. The s20,w of 5.8 S and D20,w of 51 μm2/s correlate with the molecular weight determined by sedimentation equilibrium. Sedimentation equilibrium experiments indicate a tendency for the enzyme to aggregate on increase in pH or on storage of the enzyme. The aggregates represent an association between six enzyme molecules. 6-Phosphogluconate dehydrogenase was dissociated to subunits of 52000 molecular weight using maleic anhydride or dodecylsulphate-polyacrylamide electrophoresis, indicating that the native enzyme is a dimer. After treatment with maleic anhydride detailed analysis of sedimentation equilibrium data indicates the presence of hexamers in addition to the monomers produced by dissociation. The correspondence between amino-acid composition, subunit size and subunit structure of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase is discussed.

COMPUTER ANALYSIS OF SEDIMENTATION EQUILIBRIUM DATA FROM PAUCIDISPERSE AND INTERACTING SYSTEMS.

The equation of Rinde is used as the basis for analysis of sedimentation equilibrium data from paucidisperse and interacting systems. Treatment of data by means of a digital computer makes it practical to treat all the data from a sedimentation equilibrium experiment in which a Rayleigh interference optical system is used, i.e., the total concentration and radial distance corresponding to each fringe. Results of sedimentation equilibrium experiments on ovine interstitial cell-stimulating hormone illustrate the method of analysis for the case where a single macromolecula solute exists in several states of aggregation which may or may not be in reversible equilibrium. The data are first treated as if they represent a paucidisperse, non-interacting system in order to illustrate the calculation of a molecular weight distribution for such a system. Subsequently, the data are treated in terms of a reversibly associating system. An argument is presented that, under the conditions customarily chosen for sedimentation equilibrium experiments, the molecular weight distribution of the entire sample calculated from results at sedimentation equilibrium is very nearly the same as at rest.