Hi
I wonder what is the mechanism by which HSc 70 Chaperons keep the nascent polypeptide chain in unfolded state before its entry to mitochondrial translocon (TOM-Translocon of Outer Mitocondria) ?
Regards
Tarun
On Tue, 02 May 2006 proteins-request at oat.bio.indiana.edu wrote :
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>>>Today's Topics:
>> 1. Re: Why Doesn't Hemoglobin Lose its color when heat like
> myoglobin does? (rekuci at gmail.com)
> 2. Re: why cant we say chaperone an enzyme? (Dr Engelbert Buxbaum)
> 3. Re: Protein Crashing out of Solution (Dr Engelbert Buxbaum)
>>>----------------------------------------------------------------------
>>Message: 1
>Date: 1 May 2006 16:19:15 -0700
> From: rekuci at gmail.com>Subject: [Protein-analysis] Re: Why Doesn't Hemoglobin Lose its color
> when heat like myoglobin does?
>To: proteins at net.bio.net>Message-ID: <1146268690.113792.327630 at e56g2000cwe.googlegroups.com>
>Content-Type: text/plain; charset="iso-8859-1"
>>> > What if the temperature is just high enough to completely denature the
> > hemoglobin? Will the red be lost?
>>The heme becomes slightly brownish when it's oxidized.
>>>>------------------------------
>>Message: 2
>Date: Tue, 02 May 2006 14:47:28 +0200
> From: Dr Engelbert Buxbaum <engelbert_buxbaum at hotmail.com>
>Subject: [Protein-analysis] Re: why cant we say chaperone an enzyme?
>To: proteins at net.bio.net>Message-ID: <e37kd2$gh9$03$1 at news.t-online.com>
>Content-Type: text/plain; charset=iso-8859-2
>>DK wrote:
>> > Partially folded states are in the equilibrium. By stabilizing one
> > of them, chaperones can be said to "catalyze" unfolding. The only
> > difference with chaperonines is an extra energy energy usage and
> > the overall efficiency of the process. By way of analogy -
> > chaperone is a hammer and chaperonine is a pressure-driven
> > nail gun. Both "catalyze" nail going in because, formally speaking,
> > the nail (when positioned properly) would, eventually, get in by
> > itself, just by gravity - only it would take too long :-)
>>But you see, chaperones do not increase the speed of folding by
>catalysing the folding process, they increase the yield by preventing
>miss-folding. The high protein coincentration in the cell makes
>aggregation a much more probable event than folding, by preventing
>aggregation chaperones make the folding process possible, yet they do
>not speed it up. A similar effect can be achieved - at least in
>principle - by dilution. Indeed, that is the principle of chaperonins:
>By putting their substrates into an Anfinsen cage they allow folding
>under infinite dilution.
>>> > > The concepts of prevention of miss-folding (both
> > >chaperones and chaperonins), of unfolding miss-folded proteins
> > >(chaperonins only) and of catalysing folding (neither) need to be
> > >carefully distinguished.
> >
> > I don't understand the principal distinction you make between
> > chaperones and chaperonines. The latter are just a subset of the former.
> > Essentially a more complexed and evolved way of doing the same
> > thing.
>>No, by definition chaperonins can use the energy of ATP-hydrolysis to
>actively unfold miss-folded proteins, chaperones can not do that. This
>is the reason why two different words were coined for the thingies.
>> > >Yes, of course there is no doubt that protein disulphide isomerases (or
> > >peptidyl prolyl cis/trans-isomerases for that matter) are enzymes, but
> > >they are no chaperones.
> >
> > I wasn't talking about isomerization at all. I brought up Cys-Cys just to
> > make purists happy: chemical catalysis involves chemical reaction
> > which means change in covalent bonds. Thus, any chaperon that
> > catalyze protein folding in a way that two Cys become stabilized
> > opposing each other and then dimerize spontaneously. Which means
> > that the chaperone did catalyze chemical reaction after all :-)
>>That is a hen-and-egg question which would have to be answered for each
>protein separately: does a disulphide bond form because two Cys come
>next to each other after folding, or is folding made more efficient
>after stabilisation of gross structure by a disulphide bond. In any
>case, disulphide bond formation is catalysed by PDIs, which are proper
>enzymes.
>> > As you can see, the concepts developed for small molecules lose
> > their strict clarity whenever large polymer with their qualitatively new
> > set of phenomena is considered. Boundaries blur and it's no longer
> > never easy to what is and what is not an enzyme or a chemical reaction.
>>No, there are really no entirely new concepts, even hydrogen bonds
>between molecules exist in small molecules and have structural
>significance, water is a case in point.
>> > >A chaperone may hold the client protein for the
> > >PDI or PPI to work on (Hsp90 or Bip for example), it is then like a lath
> > >on which different tools may be used. But the actual work is done by the
> > >tool, not the lath.
> >
> > The actual work is done by both. Case in point: enzymatic catalysis
> > is done by *proteins*, not just their active sites.
>>This is not an appropriate comparison. Here you have two different
>entities with different purposes: An enzyme which catalyses bond
>formation (PPI, PDI) and a chaperone that prevents things from going in
>the wrong direction. One helps in forming a bond, the other prevents the
>formation of unwanted bonds. These are two different concepts, whether
>you see the difference or not. And that is the reason why the scientific
>community decided that chaperones are not considered enzymes, at least
>with respect to the folding process (ATPase and transport activities
>are, as discussed, enzymatic processes).
>>>>>------------------------------
>>Message: 3
>Date: Tue, 02 May 2006 14:47:33 +0200
> From: Dr Engelbert Buxbaum <engelbert_buxbaum at hotmail.com>
>Subject: [Protein-analysis] Re: Protein Crashing out of Solution
>To: proteins at net.bio.net>Message-ID: <e37kd6$i1$00$1 at news.t-online.com>
>Content-Type: text/plain; charset=iso-8859-2
>>Kristina Blake wrote:
>> >
> > Hi All-
> > I am trying to purify a protein from Insect Cells (SF9) and
> > everything works perfectly, clean protein, good yield etc., until I do a
> > dialysis step into the actual buffer I need to use for my experiments.
> > The purification/elution buffers are all 20 mM Tris plus 100 mM KCl
> > pH8.0 and then I need to switch to a PIPES buffer 100 mM KCl, 2 mM
> > EGTA, 1 mM MgSO4, and Brij-35 0.25% pH6.8. During this overnight
> > dialysis is when the protein crashes, when I take the dialysis cassette
> > out the next day I can actaully see "blobs" of protein!
> > I need to switch into the PIPES buffer since I will be doing
> > protein/microtubule assays and microtubules "like" the PIPES buffer.
> > Also I've never seen the assays performed with more then 100mM salt or
> > more then 0.25% detergent presummably becasue people feel this would
> > disrupt the microtubules.
>>Do you have any detergent in your isolation buffer? If you go from high
>to low detergent, or from one detergent to another, precipitation may
>occur.
>>Also, if you have a negatively charged detergent like SDS in your
>isolation buffer and add bivalent cations like Mg, the detergent will
>precipitate and may or may not take the protein down as well.
>>Check wether Brij is actually dialysable
>(http://psyche.uthct.edu/shaun/SBlack/detergnt.html), I am currently
>offline. Also check that your 0.35% are above the cmc.
>>Could the pH of your PIPES buffer be close to the pI of your protein
>(isoelectric focussing will tell you)? That too would reduce the
>solubility of your protein.
>>Mg might precipitate proteins, although that's rare.
>>The problem is that you are changing several factors at once, so it is
>difficult to pinpoint the problem. Do small size experiments where you
>only change one factor (pH, buffering ion, detergent, bivalent metal).
>>>------------------------------
>>_______________________________________________
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>***************************************
Tarun Gupta
B.Sc.(hons)Bioinformatics
GGDSD College of Post Graduation
(Panjab University)
Sector-32/C
Chandigarh-160031
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