On 10-05-10 08:03 AM, Sudhir Kumar wrote:
> hi all
> this is just a basic query or rather for discussion.
> 1. What maintains the active state of the protein during
> crystallization under different condition which is altogether
> different condition from what that protein might be in vivo?
>
The same forces that maintain a protein in solution also maintain it in
the crystal. Protein-protein interactions that keep the crystal together
are rather weak and tend to only/mostly affect parts of the protein that
are already flexible. In other words, if a part of a protein adopts
multiple conformations, a crystal interactions may stabilize one of
them. You loose the information about the flexibility but the structure
you get is still one of the "natural conformations. Different solution
conditions can affect structure and you can find examples of pH induced
changes and probably others. But again experience shows that proteins
retain their structures through a wide range of conditions, or perhaps
conditions that mess up the structure simply never crystallize.
> 2. what is the probability of a nonfunctinal state of a protein
> getting crystallize?
>
Proteins that have a nonfunctional state in solution because they need
to be activated, proteolytically cleaved, etc. can be crystallized in
that state. Proteins that occur in open and closed states may be pushed
to the closed form by the precipitating agents and if this may
correspond to an inactive state. One of my projects had crystals of a
hemocyanin grown at high NaCl concentrations. Cl- is a known allosteric
inhibitor and it locked the protein in the low affinity state. Another
project in the lab involved a thioredoxin where the active site cysteine
became inactive through arsenylation due to the use of cacodylate
(dimethyl-arsenate). So if you search for it there are examples, but in
many cases the inactive form still represents one of the physiological
relevant forms.
> 3. Is crystal structure the actual structure of the macromolecule or
> is it rather near-actual structure?
>
Perhaps the better way to look at it is that proteins do not have one
"actual structure". They are flexible molecules that can adopt a
multitude of slightly, and sometimes not so slightly, different
structures. The core features tend to be well defined but the atomic
motions that do occur are significant compared to the atomic positional
errors of crystal structures.
> i apologize if just in case this question is not upto level of discussion.
> thanks
>
no problems, I fear our nice atom model images tend to make people
forget that proteins are not static.
Bart
--
===========================================================================
Bart Hazes (Associate Professor)
Dept. of Medical Microbiology& Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone: 1-780-492-0042
fax: 1-780-492-7521
===========================================================================
|
