Ultracentrifugation Pervushin

Analytical ultracentrifugation techniques

Theodor Svedberg invented the analytical ultracentrifuge in 1923, and won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge.

Acceleration up 1,000,000 g (9,800 km/s²).

Protein Science (2002), 11:2067–2079.JACOB LEBOWITZ, MARC S. LEWIS, AND PETER SCHUCK

Sedimentation velocity and equilibrium

Sedimentation velocity

Svedberg equation

Svedberg equationThe sedimentation velocity coefficients for smooth compact spherical proteins in water at 20°C.

The program SEDNTERP (http://www.rasmb.bbri.org)calculates Ssphere/Sexp

SEDNTERP calculates partial specific volume v from the amino acidcomposition of the protein

If the experimental Sexp is significantly higher than Ssphere,

one can conclude that the quaternary state is not monomeric,whereas a lower value would indicate an extended shape ofthe monomer caused by a larger frictional coefficient.

Example of 50 kDa protein with v of 0.730 mL/g

At 20oC in water Sexp = 5.87 S

calculated Ssphere = 4.43 S

Self-association

calculated Ssphere (dimer) = 7.77 S f/f0 would be 1.33, which supports the formation of a dimer with a moderately extended shape.

Sedimentation equilibrium

Sedimentation equilibrium

Characterization of membrane proteinsusing sedimentation equilibrium

A molar mass determination by SE would yield the sum of the protein mass and the mass of bound detergent.

The buoyant molar mass of a protein–detergent complex can be decomposed into a term for the protein and a term for the detergent, respectively

Density matching

Reynolds and Tanford (1976)

Solution NMR methods

Solution NMR of tumbling molecules

Spin relaxation

Multidimensional NMRMultidimensional NMR

Use of NOESY for 3D structure reconstructionUse of NOESY for 3D structure reconstruction

1Hppm

1H ppm 09

0

9

Heme Chaperone

3D Spectrum3D Spectrum