Molecular weight determination of ClpB by analytical centrifugation

1

• Molecular weight determination of ClpB by
  analytical centrifugation

2

• Molecular weight determination of ClpB by
  analytical centrifugation
• Biochemistry, 2003, 42, 14242-48, Kedzierska et
  al.
• ClpB is a member of the AAA family of ATPases.
  Many other members form 7-membered rings, but
  ClpB seems to form monomers (the tested
  conditions). This was tested with analytical
  centrifugation.

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• Molecular weight determination of ClpB by
  analytical centrifugation

4

• Molecular weight determination of ClpB by
  analytical centrifugation
• In the centrifuge the heavier protein molecules
  (r 1,35 g/cm3) goes outwards where their
  potential energy is lower
• But not all protein can go to the bottom of the
  tube because diffusion will push it back to the
  lower concentration (G RTln(c))

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• Potential energy
• w angular velocity (Winkelgeschw., 1 rpm
  2p/60 s-1)
• v specific volume (1/rP)
• can be calculated from amino acid
  composition
• rS density of the solvent
• Chemical potential

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• The total energy of the protein is constant
• Equilibrium (the difference of the chemical
  potential at r2 and r1 is the same for potential
  and chemical energy)
• Use r1 as the reference radius r0 and r2 as a
  general radius r and resolve

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• Fit to the radius dependent absorption
  (concentration) curve

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• Fit to the radius dependent absorption
  (concentration) curve
• s 2.02 and 2.47 M 20300 and 24900 g/mol

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• Fitting to the original data (Abs) is better than
  fitting to the linearized form ln(c(r2)/c(r1)),
  because the relative error for the absorption at
  small radii is larger