Ultracentrifugation Pervushin
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Transcript of Ultracentrifugation Pervushin
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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
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Sedimentation velocity and equilibrium
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Sedimentation velocity
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Svedberg equation
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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.
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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.
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Sedimentation equilibrium
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Sedimentation equilibrium
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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)
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Solution NMR methods
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Solution NMR of tumbling molecules
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Spin relaxation
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Multidimensional NMRMultidimensional NMR
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Use of NOESY for 3D structure reconstructionUse of NOESY for 3D structure reconstruction
1Hppm
1H ppm 09
0
9
Heme Chaperone
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3D Spectrum3D Spectrum