From: Arthur Rowe <arthur.rowe@nottingham.ac.uk>
  To  : hlashuel <hlashuel@picower.edu>
  Date: Thu, 22 Mar 2001 13:19:05 +0000

Re: A trimer with a central channel

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Hilal -
A central channel within a trimeric structure will probably not, in itself,
have too large an effect on sedimentation properties. I bear in mind
apoferritin, a protein with a very well defined central cavity, whose
hydrodynamics - give or take a slightly elevated frictional ratio - are
reassuringly normal. Including its vbar value - which we have ourselves
studied in some detail. So, at most you will get a slightly lower s value
than expected for a molecule of whatever size it is.
However, it could well be that in solution a structure which is loosely
packed might adopt a range of closely related structures, each with its own
frictional ratio.  This would give a spreading of the boundary in
sedimentation velocity, which if attributed to diffusion would result in to=
o
large a value for the diffusion coefficient being yielded by whatever
fitting algorithm was employed.  From the which, of course, too low an
estimate for M would result from combining an average s with an aberrant D
value.  Sedimentation equilibrium, however, is unaffected by shape - at
least in dilute solution, so values of M from that method should be OK.
Looking at your values, things are a bit puzzling.  Treating your protein a=
s
a 'typical globular protein' and estimating M from the s value alone, the
vbar being treated as unknown (what is it ?), the equations of Squire &
Himmel, based upon a pretty massive compendium of experimental data, give a
value of M =3D 34531 =B1 3107.  Which is compatible with a trimer, but not with
a dimer.
Going a bit further, if we assume a trimer, vbar =3D 0.73 ml/g, then f/f0
(frictional ratio) =3D 1.37, which sounds not too unreasonable in the light o=
f
that central cavity (see para 1 above). However, if we force it to be a
dimer, then we get f/f0 =3D 1.04, which sounds distinctly small - although I
guess there is no cavity to be accounted for in a dimer. The weight-average=
d
s value should be solid, so on the face of it a trimer sounds probable.
BUT - those M values from SE of around 27 kD - how to explain ?  They do
suggest that maybe some sort of equilibrium is present, and that SE - with =
M
values maybe derived from the near-meniscus end of an intermediate/high
speed run (?) - giving a low-ish impression ?  If the trimer is loose in
structure, perhaps you might have 2xtrimer in equilibrium with 3xdimer
(intermediates short lived) ?
Fascinating system - all the best with it !
Arthur Rowe
*****************************************************
Arthur J Rowe
Professor of Biomolecular Technology
University of Nottingham
School of Biological Sciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Phone/voicemail       +44 (0)115 951 6156
Phone/fax             +44 (0)115 951 6156/7
email                 arthur.rowe@nottingham.ac.uk
                      arthur.rowe@connectfree.co.uk
Web                   http://www.nottingham.ac.uk/ncmh/business
*****************************************************

----------
>From: hlashuel <hlashuel@picower.edu>
>To: "rasmb@rasmb.bbri.org" <rasmb@alpha.bbri.org>
>Subject: A trimer with a central channel
>Date: Sun, Mar 21, 2004, 11:57 pm
>

>I am currently working with a protein that is 12.4 KDa in MW and 
>crystallizes as a symmetric trimer. Association of the three monomers 
>results in the formation of a solvent accessible channel that runs 
>through the molecule giving it an unusual donut shape. The solution 
>characterization of this protein has been controversial. gel-filtration, 
>NMR and sedimentation equilibrium (rat protein) of this protein have 
>reported a dimer in solution. However, cross linking studies have 
>suggested a mixture of monomer, dimer and trimer. Sedimentation velocity 
>studies carried out in our laboratory show that this molecule sediments 
>as a single species with an s value of 2.94 S and a molecular weight of 
>24-25 KDa. However, sedimentation equilibrium yielded a MW of 27 KDa. I 
>would like to know what everyone thinks about the central channel of this 
>protein, and how it might effect the hydrodynamic properties of this 
>protein. Can a trimer (37 KDa behaves like a dimer (24.7 KDa) in 
>solution. This proteins elutes as three peaks in gel filtration exp, but 
>the data could not be fitted to a monomer, dimer and trimer model. 
>Sedimentation equilibrium carried out for each fraction revealed a MW of 
>26-27 KDa for each peak. NO monomer could be detected.
>
>
>
>your comments will be highly appreciated. 
>
>
>Hilal



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<HEAD>
<TITLE>Re: A trimer with a central channel</TITLE>
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<FONT SIZE=3D"2">Hilal -<BR>
A central channel within a trimeric structure will probably not, in itself,=
 have too large an effect on sedimentation properties. I bear in mind apofer=
ritin, a protein with a very well defined central cavity, whose hydrodynamic=
s - give or take a slightly elevated frictional ratio - are reassuringly nor=
mal. Including its vbar value - which we have ourselves studied in some deta=
il. So, at most you will get a slightly lower s value than expected for a mo=
lecule of whatever size it is.<BR>
However, it could well be that in solution a structure which is loosely pac=
ked might adopt a range of closely related structures, each with its own fri=
ctional ratio.  This would give a spreading of the boundary in sedimentation=
 velocity, which if attributed to </FONT><U>diffusion</U><FONT SIZE=3D"2"> wou=
ld result in too large a value for the diffusion coefficient being yielded b=
y whatever fitting algorithm was employed.  From the which, of course, too <=
/FONT><U>low</U><FONT SIZE=3D"2"> an estimate for M would result from combinin=
g an average s with an aberrant D value.  Sedimentation equilibrium, however=
, is unaffected by shape - at least in dilute solution, so values of M from =
that method should be OK.<BR>
Looking at your values, things </FONT><U>are</U><FONT SIZE=3D"2"> a bit puzzl=
ing.  Treating your protein as a 'typical globular protein' and estimating M=
 from the s value alone, the vbar being treated as unknown (what is it ?), t=
he equations of Squire & Himmel, based upon a pretty massive compendium =
of experimental data, give a value of M =3D 34531 =B1 3107.  Which is compatible=
 with a trimer, but </FONT><U>not</U><FONT SIZE=3D"2"> with a dimer.<BR>
Going a bit further, if we assume a trimer, vbar =3D 0.73 ml/g, then f/f0 (fr=
ictional ratio) =3D 1.37, which sounds not too unreasonable in the light of th=
at central cavity (see para 1 above). However, if we force it to be a dimer,=
 then we get f/f0 =3D 1.04, which sounds distinctly small - although I guess t=
here is no cavity to be accounted for in a dimer. The weight-averaged s valu=
e should be solid, so on the face of it a trimer sounds probable.<BR>
BUT - those M values from SE of around 27 kD - how to explain ?  They do su=
ggest that maybe some sort of equilibrium is present, and that SE - with M v=
alues maybe derived from the near-meniscus end of an intermediate/high speed=
 run (?) - giving a low-ish impression ?  If the trimer is loose in structur=
e, perhaps you might have 2xtrimer in equilibrium with 3xdimer (intermediate=
s short lived) ?<BR>
Fascinating system - all the best with it !<BR>
Arthur Rowe<BR>
*****************************************************<BR>
Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
University of Nottingham<BR>
School of Biological Sciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD   UK<BR>
<BR>
Phone/voicemail       +44 (0)115 951 6156<BR>
Phone/fax             +44 (0)115 951 6156/7<BR>
email                 arthur.rowe@nottingham.ac.uk<BR>
                      arthur.rowe@connectfree.co.uk<BR>
Web                   http://www.nottingham.ac.uk/ncmh/business<BR>
*****************************************************<BR>
<BR>
----------<BR>
>From: hlashuel <hlashuel@picower.edu><BR>
>To: "rasmb@rasmb.bbri.org" <rasmb@alpha.bbri.org><BR>
>Subject: A trimer with a central channel<BR>
>Date: Sun, Mar 21, 2004, 11:57 pm<BR>
><BR>
<BR>
>I am currently working with a protein that is 12.4 KDa in MW and <BR>
>crystallizes as a symmetric trimer. Association of the three monomers <=
BR>
>results in the formation of a solvent accessible channel that runs <BR>
>through the molecule giving it an unusual donut shape. The solution <BR=
>
>characterization of this protein has been controversial. gel-filtration=
, <BR>
>NMR and sedimentation equilibrium (rat protein) of this protein have <B=
R>
>reported a dimer in solution. However, cross linking studies have <BR>
>suggested a mixture of monomer, dimer and trimer. Sedimentation velocit=
y <BR>
>studies carried out in our laboratory show that this molecule sediments=
 <BR>
>as a single species with an s value of 2.94 S and a molecular weight of=
 <BR>
>24-25 KDa. However, sedimentation equilibrium yielded a MW of 27 KDa. I=
 <BR>
>would like to know what everyone thinks about the central channel of th=
is <BR>
>protein, and how it might effect the hydrodynamic properties of this <B=
R>
>protein. Can a trimer (37 KDa behaves like a dimer (24.7 KDa) in <BR>
>solution. This proteins elutes as three peaks in gel filtration exp, bu=
t <BR>
>the data could not be fitted to a monomer, dimer and trimer model. <BR>
>Sedimentation equilibrium carried out for each fraction revealed a MW o=
f <BR>
>26-27 KDa for each peak. NO monomer could be detected.<BR>
><BR>
><BR>
><BR>
>your comments will be highly appreciated. <BR>
><BR>
><BR>
>Hilal<BR>
<BR>
<BR>
</FONT>
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