[RASMB] problem with fitting

Thu Mar 20 09:29:09 PDT 2008

Hello Marina,

As I try to clean up my old e-mail and notice that I miss this
discussion about two weeks ago.   I remember I did an experiment when
Sedfit was release.  The reason for doing this experiment is when I
analyzed the sample with glycerol presence in the buffer, the S value is
always smaller than when I analysis the data using DCDT +, even when I
put in the correct density and viscosity value.  So I derived an
experiment using CRP protein (a dimmer protein with molecular weight
about 47KDa) in the presence of different concentration of glycerol. I
am sending this in the attachment.  If I use the correction constant,
then your protein of 6.23S should really be 1.21X 6.23=7.54 S. This will
reconfirm it is a trimmer but will not change the trimmer to hexamer as
you expected. I hope this information is useful for you.

All the best,

Christopher Chin

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Thomas Jowitt
Sent: Monday, March 03, 2008 4:39 AM
To: thomas.a.jowitt at manchester.ac.uk; Fasolini, Marina [Nervianoms];
rasmb at rasmb.bbri.org
Subject: RE: [RASMB] problem with fitting

Hi Marina

Just a follow up to my previous reply. I notice you are running the
sample in 5% glycerol? The viscosity of this alone is 1.127E-2, not what
you have mentioned in your mail. If this is the case then this would
alter the S20,w of your protein substantially. 

Thanks

Tom

________________________________

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Thomas Jowitt
Sent: 03 March 2008 10:17
To: Fasolini, Marina [Nervianoms]; rasmb at rasmb.bbri.org
Subject: RE: [RASMB] problem with fitting

Dear Marina

First of all, unless your gel filtration is linked to a light scattering
module to estimate the absolute molecular mass, then I wouldn't trust
the estimated molecular weight from a calibrated column. A sedimentation
value of 6.23 is most likely to be around 150kDa unless the molecule is
substantially elongated, as a hexameric molecule of this size and
sedimentation value would have a frictional ratio of around 2.5
(certainly not impossible). 

The two experiments that I would perform to answer these questions are,
firstly an equilibrium experiment to establish the correct molecular
weight, done with at-least three concentrations to establish if
oligomerisation is concentration dependant. Secondly, I would run the
crystal structure through a modeling program such as SOMO to generate a
bead model, or hydropro to create a shell around the crystal structure.
Either of these would give you a decent estimate of the sedimentation
value expected for a rigid molecule of that size. 

Thanks

Tom Jowitt

________________________________

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Fasolini, Marina [Nervianoms]
Sent: 03 March 2008 09:10
To: rasmb at rasmb.bbri.org
Subject: [RASMB] problem with fitting

Dear AUC users,

I have a problem in the fitting of some data and I would like to have
your opinion.

I want to control the sedimentation of a protein in order to define the
state of oligomerization. It is published as forming an  hexameric homo
oligomer. From the crystal structure, it is a wheel like structure of
160 angstrom  in diameter, with a central role of 15 angstrom in
diameter. The thickness is 20-40 angstrom. How can I expect it to
sediment? How can I improve my parameters or model in order to improve
the fitting?

The protein is 50KDa  but in gel filtration it cames out as  hexamer
(300 KDa). In my sedimentation experiment I see one peak of  6.23S which
corresponds to 140KDa. Fitting of the sedimentation data was done with
SedFit. Do you think I can say that it is a trimer? I would expect it as
an hexamer.

I hope someone can help me. 

the conditions are : 

buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly 

Vbar 0.7397 

Viscosity 1.02530 

Density 0.01183 

Thanks a lot

Marina 

MARINA FASOLINI

Structural Chemistry 

Nerviano Medical Sciences <http://www.nervianoms.com/> 

Viale Pasteur 10

20014 Nerviano - Milano

marina.fasolini at nervianoms.com

Tel. +390331581462

Fax. +390331581360

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