[RASMB] aggregates data analysis

[Tom Laue]

Hi Joris-
The lack of a gradient at the meniscus indicates that there is a fairly 
strong association to larger species. The lack of an exponential 
distribution suggests that the system is exhibiting very strong 
repulsive nonideality.  You are working at low ionic strength, and have 
a reduced dielectric constant due to the methanol. The nonideality is 
most likely due to charge-charge repulsion. There is no way excluded 
volume effects would be this strong. I would be suspicious of a primary 
charge effect, but the lack of a gradient in the meniscus region argues 
against this being significant.
Since the monomer is only 4 kDa, and you have meniscus depletion at 30 
k, the stoichiometry of assembly is quite large... 50, 100... depending 
on vbar. Unless the nonideality is accounted for, you will have 
difficulty fitting this.
Best wishes,
Tom

Joris Beld wrote:
> Dear all,
> Sorry for not replying earlier. First a few remarks:
> - the 'take off' point goes down the cell at higher speeds
> - 30krpm was too fast but since the monomeric species is only 4kDa and 
> we had no idea about the aggregating state we chose as a first speed 
> 30krpm
> - Thanks to Allen Minton for the reprint! This looks similar to what 
> we observe.
> - The ionic strength of the solution is low (10mM) and it's a 10% 
> methanol/water solution
> - ultrascan: I used the single-ideal-species model to fit the data as 
> well as the fixed MW model (from 10k-1MDa) and both do not fit well 
> since the curvature is very non-ideal
>
> I have attached an example of a scan at equilibrium (two days) at 
> 10krpm. The data is not great. Data has been obtained at 260nm, 20C, 
> 120ul of liquid column and 30ul of FC43.
>
> For me it seems like it could be two things:
> - either this peptide/protein aggregates heterogeneously and forms a 
> whole bunch of different sized aggregates (which would make sense with 
> -for example- our CD measurements and it would make sense with the 
> designed characteristics of this peptide/protein)
> - or the conditions are so non-ideal with the low salt concentration 
> and the methanol present that this influences the behavior too strongly.
> I'm running now a SV run to see whether this behavior is also seen in 
> this experiment.
> Thanks a lot for the help.
> Best,
>
> Joris
>
>
>
> Leech, AP wrote:
>> Hello Joris,
>>
>> I would expect the "take off point" to move down the cell at higher
>> speed. Does this happen? Do you have scans for the approach to
>> equilibrium to see how this distribution develops?
>>
>> For 280 kDa and 30000 rpm I would also expect everything to be pelleted
>> at the bottom. Do you have an expected Mw for the sample?
>>
>> It might be useful to post a plot of the data. If it is not some sort
>> of artefact then maybe a velocity experiment would be informative.
>>
>> Best regards,
>>
>> Andrew
>>
>>
>> Beld, Joris wrote:
>>> Dear all,
>>>
>>> I'm spinning a sample which shows -to me- surprising behavior. The 
>>> raw data of a normal sedimentation equilibrium run of a protein 
>>> looks always like a half-parabolic curve of the radius versus 
>>> absorbance (x,y).
>>> However, this particular sample (protein) shows a different behavior 
>>> in that the raw data look like a straight line taking off halfway 
>>> the liquid column (absorbance almost zero) in the sector to the 
>>> bottom of the cell (absorbance 0.7). Same behavior is reproducible 
>>> at three different concentrations and three different rotational 
>>> speeds (10,20,30 krpm).
>>> How should I interpret these data? Ultrascan does not really 'like' 
>>> this data as input (although fitting is possible, the residuals look 
>>> very bad, the fitted Mw -to a single species- is around 280kDa).
>>> Is this how a heterogeneous aggregating sample should look like?
>>> Thanks in advance.
>>>
>>> Best wishes,
>>>
>>> Joris Beld
>>> ETH Zürich
>>> Switzerland
>>> _______________________________________________
>>> RASMB mailing list
>>> RASMB at rasmb.bbri.org
>>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>>>
>>
>
>
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Department of Biochemistry and Molecular Biology
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E-mail: Tom.Laue at unh.edu
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