Prob. 2-5 part C, "Would urea reduce the molecular weight
of a protein composed of more than one polypeptide chains)?"
The answer provided is yes, my first response was no.
However, I am thinking of why this may be so... could it be
that urea may cause the separation of one polypeptide which
would alter the MW of a polypeptide protein?
Say the MW of a tetrameric protein (i.e., a protein that has quaternary
structure) is 64000 (e.g., Hb). Treat with urea. The 4 monomer
polypeptides come apart. Now if you determine the MW (of the subunits),
it is 16000. A protein is defined as the whole shebang, all the
subunits together if there is quaternary structure. All this assumes no
disulfides between the subunits.
How does treatment with urea change the MW of a protein if urea just disrupts
weak bonds & interactions? I thought urea only changed quaternary, tertiary
and secondary structure.
Because urea can disrupt quaternary structure, it can conver a native
multi-subunit protein to its constituent subunits (as long as there are no
disulfide bonds holding the subuints
together). The subunit will have lower MWs than the the intact native protein.
For problem 2-14 in the coursepack, I was under the impression that the
polypeptide chain, although denatured in urea would fold back into its original
position upon removal of the urea. The answer key however states that it will
not refold itself the same way (ie. removal of urea does not rejoin bonds). I
thought this was a similar question to the Anfisen experiment which proved that
primary structure determine tertiary structure. Can you please explain why the
bonds will not rejoin in problem 2-14. Would this be the case with all
chaotropic agents?
In 2-14, the point in this made-up example is that the ribonuclease DOES return
to its original 3-dimensional conformation, except for the reformation of the
one covalent bond, the peptide bond, that was cleaved. All the weak bonds
(non-covalent bonds), that are so important in holding the molecule in its
distinct tertiary structure have returned. In a way, the protein has been
converted from a single polypeptide protein to a multimer of 2 subunits, held
together by weak bonds, and whose 3-dimensional structure is essentially the
same as when that peptide
bond was still there. So this example is sort of a super-Anfinsen experiment,
renaturation was achieved even though a a covalent peptide bond was broken and
not restored. Denaturation-renaturation never involves breaking or reformation
of peptide bond; the only covalent bond allowed to break in
denaturation-renaturation is the disulfide.
Does urea only act on hydrogen bonds by competing with the
ability of
side chains to h-bond? If so, how does it affect, or does it affect, ionic,
VDW and hydrophobic interactions?
The exact nature of urea dentaturation is not understood, as far as I know.
Because of its ability to H-bond extensively, it is likely to disrupt H-bonds
and ionic bonds.