Summary: Protein dimerisation in SDS gels

[Lutz Essers]

Hi everyone,

In the end of January I have asked about dimerisation of proteins in SDS 
gel. Here I want to give a short summary about the answers you gave me 
during the last days.

First: YES several people have reported about proteins, which seems to 
form dimeres or oligomers in SDS gels!


WHAT KINDS OF PROTEINS SHOW DIMER FORMATION IN SDS GELS?

I think, it is not easy to find out a common property.
- Most of the reported proteins have transmembrane domains, can bind to 
membranes or fatty acids.
- Some of the reported proteins are relative small, although I was not 
able to check it in each case (relative small means < 15-20 kd)
- There are examples of proteins which "are composed mainly of 
beta-structure" and of proteins, where "no beta structures is present" 
("... it's all-alfa helixes").

WHAT CAN BE THE REASONS FOR SDS / BETA-MERCAPTOETHANOS RESITENCE OF
DIMER FORMATION?
CAN THE DIMER FORMATION IN SDS GELS BE SUPPRESSED?

No common reason for dimerisation in the presence of SDS is known. 
Instead of this there are hints, which may indicate distinguishable 
processes.

In the following I have made one speculative classification of reasons 
with some reported tricks to suppress dimer formation. These tricks may 
work or not - depending on the nature of each protein.


1) the proteins are not denaturated well enough and can still 
multimerise

This may be supported by the fact that proteins can dimerise although 
they do not contain cysteins. 
The partial denaturation may be especially the case for smaller 
proteins. Long time ago I have read that e.g. in the average one 
SDS-molecules binds per about 40 amino acids (is that right?).

-> Use SDS gels with aditional 6 M urea.
-> Boil your samples. Some people said, that their dimers are resitant 
to SDS and 8 M urea, but comes apart when boiled.


2) not all intermolecular disulfide bonds are broken

Yes, disulfide bonds, that is what we learn in the school ;-)
The dimerisation of several proteins was reported to be depending on the 
presence of cystein.

-> Pretreatment with b- mercaptoethanol normaly suppressed the 
dimerization
-> Mix also an amount of b-ME to the gel
-> Use 25 mM DTT instead of b-ME
-> Again: Some people said, that their dimers are resitant to SDS but 
comes apart when boiled. (Did they mean boiling with b-ME or DTT?).


3) general

Some people mentioned, that they have seen oligomers only with purified 
proteins. Can this indicate, that otherwise the proteins react with 
other proteins in an unspecific way (->smear?), so that no clear 
oligobands can be seen?

-> do not purify your protein, hahahahaa!


4) other possibilities

There are other possibilites for dimerisation of proteins. Lauge thinks 
"that dimers in the 0.01-0.1% range can be explained by natural chemical 
crosslinking mechanisms such as transamidations".
It was also speculated that dimerisation has something to do with 
glycosylation AND high hydrophobicity of some proteins.
The following tricks *can* suppress dimerformation in gels. Maybe they 
have something to do with transamidations or the dependency of 
glycosylation.

-> Do NOT heat the protein samples in Laemmli-buffer. Instead of the 
incubate 5 minutes at 37 degree C and minimise the time from sample 
dissolution in SDS loading buffer. (I have done it. Yeah, it reduces my 
dimer bands!  Lutz)
-> Check: Boil your sample for a longer time and look, if you will get 
stronger dimer bands. This may give you a hint about your dimers.
-> Ad some sucrose to the loading buffer


ARE THERE ANY PUBLISHED COMMENTS ABOUT SDS-RESITENCE OF DIMER
FORMATION?

Look yourself to the following references. I thing some of them are 
helpfull for further answers (and questions?).

Biochemistry 1992, 31, 12719-25

Journal of Biological Chemistry vol 267, No 11, Apr 15 pp. 7683-89

Tokunaga, M et al. (1979) European  J. Biochem. 95: 441-448

Yu, F et al. (1979) FEBS Letters 100: 71-74

Griffin, T. J. and Kolodner, R. D., Purification and preliminary 
characterization of the Escherichia coli K-12 RecF protein, Journal of 
Bacteriology, 172, 6291, 1990.

PNAS 90: 3840-3844

Virology 203:320-327

MANY THANKS TO ALL WHO HAVE PARTICIPATED TO THIS DISCUSSION:

     Tom Bickle
     Anthony A. Bominaar
     Albert Haid
     Dima Klenchin
     John Marcus
     Mick Partis
     Qiudeng Que
     Alberto I. Roca
     Lauge Schaffer
     Andrey Shaw
     Dave Smith
     Dr.Sailesh Surapureddi
     Michael Szardenings
     Michael Vork
     Vadim

___________________________________________


Lutz Essers, Universitaet Munich
E-Mail: uj44228 at sunmail.lrz-muenchen.de

(NEW E-Mail, starting within the next days:
 Essers at gen1.genetik.uni-koeln.de)