Dear
Kushol, Chad and other folks;
Thanks a lot for the comments. I am
sorry for keeping silent because we are still repeating the SE experiment.
However,
here are some preliminary data.
Did
Jerry run SV or SE?
2. If the former, then did he see a nice, single boundary, or a big
smear?
We
tried both SV and SE. SV showed an increasing Sw with decreasing concentrations
(Sw varied from 6.9, 7.0, 7.2, 7.4 to 7.522). IN addition, the SV experiments
showed a
single boundary. However, the boundary was a little broader, between 6 and 8.5.
SV peaks seem to overlay at lower
concentrations with Sw ranging from 7.5 to 7.86. From SV experiments, the
calculated Mw is ~125
KD with f/fo equal to 1.2. The monomer of my protein is ~20KD. I am not sure
whether 7.86 S would be much faster than a tetramer (~80KD) could possibly
sediment.
In
the meanwhile, light scattering on my sample showed a radius equal to a
tetramer.
3. If the latter, how were the data analyzed to come to his
conclusion?
At present, we also want to measure the Vpar in order to better fit the SE
data. Any suggestions here?
The problem is that we only observed tetramers
in the crystals which were grown from PEG, which probably would keep the
protein’s native oligomerization state.
How could we interpret the discrepancy?
Thanks again and welcome more comments
and suggestions.
Jerry
Date: Thu, 2 Jun 2011 11:37:57 -0700
From: cabrautc...@yahoo.com
Subject: Re: [ccp4bb] larger molecular weight shown by analytic
ultracentrifugation
To: CCP4BB@JISCMAIL.AC.UK
Dear Kushol & Jerry,
I have to take exception to Kushol's contention about SV. As long as the
buffer and protein parameters are correct and the sample is well behaved (i.e.
not undergoing dynamic rearrangement on the time scale of the SV experiment,
not aggregated, homogeneous), one can derive very accurate molar masses from
SV, and it is far superior in this respect than SEC.
However, as you aptly point out, the problem may lie in the sample's behavior.
If the protein is populating multiple oligomeric states, or if it is undergoing
a fast interconversion of such states, or if it is not pure, spurious masses
may be calculated. If such pathologies are observed, it's not clear to me that
a sedimentation equilibrium experiment would help. For a
complicated interaction model, SE data (and AUC data in general) can be
difficult to analyze, and some model assumptions are almost always present.
So, the questions are:
1. Did Jerry run SV or SE?
2. If the former, then did he see a nice, single boundary, or a big smear?
3. If the latter, how were the data analyzed to come to his conclusion?
Hopefully he can comment on these aspects.
Cheerio,
Chad
From: Kushol Gupta <kushol.gu...@gmail.com>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, May 27, 2011 9:51:04 AM
Subject: Re: [ccp4bb] larger molecular weight shown by analytic
ultracentrifugation
Hi Jerry –
By AUC, do you mean sedimentation velocity (SV)?
Both gel filtration and SV are not terribly great ways to
determine precise molecular mass, especially if the macromolecule of interest
is anisotropic in shape. In your SV values, do you see a large f/fo, or a
broad distribution? Can you run a sedimentation equilibrium experiment? If you
run HYDROPRO on your prospective oligomer structure, do you arrive at
theoretical S and Rs values that jive with your solution data?
A nice crosscheck you could do with the data in hand (if your
measurements from both approaches were performed in the same buffer at the same
temperature) is calculate the mass using the Siegel and Monty equation (Siegel,
L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346–362), where
the mass of the particle is calculated from Rs (from gel filtration) and the
S(t,b)
value from sedimentation velocity.
Hope this helps,
Kushol
Kushol Gupta, Ph.D.
Research Associate
Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of
Medicine
kgu...@mail.med.upenn.edu
215-573-7260 / 267-259-0082
From: CCP4 bulletin board
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully
Sent: Friday, May 27, 2011 10:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] larger molecular weight shown by analytic
ultracentrifugation
Dear
ALL;
I am sorry for this off-topic question about analytic
ultracentrifugation (AUC).
We recently solved one structure from crystals grown out of
PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein
would maintain its native oligomerization state as in the solution.
Indeed, the crystal packing clearly shows a tetramer of this
protein. However, both the gel-filtration and AUC showed larger molecular
weight, roughly around 6-mer or 7-mer.
IN the crystal lattice, we could not find any 6-mer or
7-mer state.
Could anyone give some comments on this discrepancy?
Thanks a lot and have a nice weekend!
Jerry McCully
