From: Jim Bloom <Jim.Bloom.B@bayer.com>
  To  : -         *rasmb@bbri.org <rasmb@bbri.org>
  Date: Mon, 8 Nov 1999 11:23:05 -0500

Re: Simulating Eq curves and Re: cavities

 Two issues, binding of water in a protein cavity and an RNA/protein
complex...problems being approached by AUC.  I was taught that there is no such
thing as a "magic bullet" technique, so might I suggest attempting mass
spectrometry?  The latest Protein Science contains an article entitled:
"Probing the nature of interactions in SH2 binding interfaces-evidence from
electrospray ionization mass spectrometry" (Chung et al. Protein Science
8:1962-1970, 1999).  In the article, the authors describe the use of nano-flow
ESI-MS to study protein/peptide binding and the incorporation of water
molecules into the protein/ligand interface.  This technique might supply
useful information to help unravel the water in the protein cavity problem
(direct measurement of masses of bound waters) as well as the RNA/protein
complex issue (depending on the accuracy of the particular ESI-MS to which you
have access, the ~75 KD would be considerably less ~ than your AUC   value ).

Jim
Bayer Corp
Berkeley, CA
Jim.Bloom.B@Bayer.Com






pjgb <pjgb@mrc-lmb.cam.ac.uk> on 11/08/99 02:16:31 AM
To: "DDA.RFC-822=rasmb@bbri.org/P=Internet/A= /C=us"@X400, edu art
<art@scripps.edu> @ INTERNET
cc:

Subject: Re: Simulating Eq curves

Dear Art,

May one enquire what partial specific volume you are using, to calculate MW
~75Kd?  In the past I have been asked to look at possible protein/RNA
ratios in a complex.  I calculated the v-bar for each possibility and used
this to calculate MW.  In practice it came out that only one ratio gave a
fit of the data to the ratio used to get v-bar.  (Incidentally this
complied with a ratio determined later by other methods.)

Jo

--On Sat, Nov 6, 1999 8:39 pm -0800 Arturo Morales <art@scripps.edu> wrote:

>
> Hi there,
>
> We have a RNA:protein system in which we are calculating a MW  ~75Kd.
> This could either represent a 2:1 RNA:protein or a 1:2 complex. (since the
> RNA and the protein monomer are both ~25Kd.  We can make an argument for a
> 1:2 complex since the 260:280 ratio appears to be closer to what we would
> expect in that type of complex (it is not quite 2:1 as we would expect for
> a complex with more RNA than protein.  We would like to model the expected
> eq curves for both possibilites using theoretical data and compare it with
> our actual data.  Altough we have a bunch of different software titles
> that can deal with velocity data, we only have the beckman stuff for
> equilibrium... Any suggestions? :)
>
> Thanks!
>
> Art
>
> ----------------------------------------------------------------------
> Arturo J. Morales    (RPI '94) |  art@scripps.edu
> Department of Biology          | Massachusetts Institute of Technology
>   Skaggs Institute for Chemical Biology - Scripps Research Institute
>                   http://minihelix.mit.edu/art
>



P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road,
Cambridge, CB2 2QH,
UK.



mail NeverRegister <kgf@mail.csb.yale.edu> on 11/07/99 04:30:13 PM
Please respond to mail NeverRegister <kgf@mail.csb.yale.edu> @ INTERNET
To: nottingham arthur.rowe1 <arthur.rowe@nottingham.ac.uk> @ INTERNET
cc: "DDA.RFC-822=RASMB@bbri.org/P=Internet/A= /C=us"@X400, biochem
j.matthews <j.matthews@biochem.usyd.edu.au> @ INTERNET

Subject: Re: cavities

Thanks to Arthur for a very thoughtful response. However, I should add that I
look forward with great interest to the results of calculations that consider
the
cavity to be entirely empty. I myself would be quite astonished if this was
actually, physically the case. Nature does after all abhor a vacuum. And in
fact, I
am not sure that I could be convinced of the existence of an entirely empty
cavity even if the crystallographers did not include, for whatever reason, any
or
all cavity waters in their model building and refinement. On the contrary, a
lack of evidence of ordered water in the cavity would only suggest to me that
the
electron density was not sufficiently precise enough to place ordered waters. I
am not a crystallographer, but it is my impression that depending on the method
and the tricks used in the crystal structure refinement, the presence of waters
in the cavity with an equivalent electron density to that of bulk solvent would
result in a minimization of their contribution to the model of the well ordered
crystallized protein.

Just my humble opinion.....

Cheers,

Karen F.

> At the other extreme, if the cavity is entirely empty, then vbar which you
calculate
> will be too small by the ratio (volume of protein incl cavity)/(volume of
protein
> residues alone).  Hence the M which you calculate will be too low, roughly by
3%
> for every 1% by which numerator in the above exceeds the denominator.
> Potentially quite a big effect.  Presumably if you have a crystal structure
you can
> make a fair estimate to the above ratio, and see what a modified vbar does to
your
> M value.  If the explanation I have given is correct (I nearly said 'holds
water' !)
> then the new M value will be either equal to or larger than the true M value.
>
> For any intermediate percentage of the cavity being filled, which you might
again
> base on your crystallographic structure, you can simply estimate a vbar by
> proportionality.  A bit of a potential problem however might be that as you
say the
> water is 'ordered', its packing density may differ from free water (as in
solvent).
> That however is a second order refinement - I suggest to try the above
calculations
> first.
>
> Best wishes
>
> Arthur Rowe
> *************************************************
> Professor Arthur J Rowe
> NCMH Business Centre
> University of Nottingham
> School of Biological Sciences
> Sutton Bonington
> Leicestershire LE12 5RD   UK
>
> phone/voicemail                                 +44 (0)115 951 6156
> phone/telefax                                                       +44
(0)115 951 6157
>
email
                                                arthur.rowe@nottingham.ac.uk
>
(home)
     arthur.rowe@connectfree.co.uk
> www.nottingham.ac.uk/ncmh/business