[RASMB] Centrifugal force and protein dissociation

[John Correia]

Concentration dependence is the key issue in determining if you have a
reversible monomer-dimer-tetramer system.  DCDT+ and Svedberg assume
noninteracting, nonreversible systems and thus if you "see" a monomer
zone, a dimer zone and a tetramer zone with fitting by DCDT+ then they
are likely to be irreversible species.  Walter Stafford showed in a
Methods Enzymol paper (vol 323?) that kinetically slow and kinetically
fast reversible systems with the same K's will all sediment with the
same weight average values, also gotten from DCDT+, but only if they
were at equilibrium at the start of the run.  A reversible 1-2-4 systems
with evolve from a monomer into a dimer-tetramer reacting boundary,
depending upon the K's, that you should be able to see as a function of
total concentration.  The weight average values should increase from
monomer to tetramer values, if you can span the correct concentrations,
and can be fitted to extract K2 and K4 values (Correia in Methods
Enzymol).    From your description I would guess you did one run and did
one fit?  Do a family of runs and plot the g(s) curves vs S* - if the
curves simply increase in height but don't move to higher S values with
concentration, evolution of the predominant tetramer species, then you
have irreversible "junk".  I also suggest repeating the run with
sufficient reductant.  We have observed reversible systems that include
aggregated species, and thus part of the zone evolves while part of the
boundary is just aggregate, in these cases often, but not always,
reductant sensitive.  The pressure dependent cases are often extremely
large polymers like myosin with large molar volumes of association - in
those cases speed dependence is the key.

I might add all of this is discussed in great detail in numerous
reviews and research articles published over the last 40 years - the
reference list is long and very informative - you should start with the
cryptic suggestions made here and go the literature for a complete
story.  A biased and partial list follows.

J.J. CORREIA (1998) "Sedimentation Velocity Data Analysis Methods:
What, When and Why?" ChemTracts, 13, 944-949.

J.J. CORREIA (2000) "The Analysis of Weight Average Sedimentation
Data." Methods in Enzymol., vol 321, 81-100.

S. Lobert and J.J. CORREIA (2000) "Energetics of Vinca Alkaloid
Interactions with Tubulin." Methods in Enzymol., vol 323, 77-103.

J.J. CORREIA, S.P. Gilbert, M.L. Moyer and K.A. Johnson  (1995)   
"Sedimentation Studies on The Kinesin Head Domain Constructs K401, K366
and K341."  Biochemistry, 34, 4898-4907.

J.J. CORREIA, B.M. Chacko, S.S. Lam and K. Lin. (2001)  "Sedimentation
Studies Reveal a Direct Role of Phosphorylation in Smad3:Smad4 Homo- and
Hetero-Trimerization." Biochemistry, 40, 1473-1482.

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 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
 fax (601) 984-1501                             
 email address: jcorreia at biochem.umsmed.edu     
 homepage location: http://biochemistry.umc.edu/correia.html  
 dept homepage location:    http://biochemistry.umc.edu
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