[urtula]

Dear all,

I am not a biochemist and lack the fundamental knowledge to really understand papers in this field.
I am struggling to understand the paper in the link: {MOD Edit: remove link}
If I understand it correctly than the hysteris mentoined in this paper means that the folding of the protein is energetically "easier" (less energy needed) than the unfolding?
thanks in advance

[Yggdrasil]

I am a bit wary of downloading the paper from your site.  Can you give a citation for the paper in question (i.e. authors, journal, year published)?

[Arkcon]

The entire paper is available with any sort of registration here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695656/

Its a lot to read, so it will be a while before I have any input on the question.  But here's the link for everybody.

[urtula]

The entire paper is available with any sort of registration here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695656/

Its a lot to read, so it will be a while before I have any input on the question.  But here's the link for everybody.

Yes thats the paper.
I used the university server and did not realise it was free.

The paper itself is pretty long, but my question if pretty basic so one might be able to answer it very fast.

I just wonder: is the unfolding of a GFP protein energetically "higher" than the folding itself due to the hysteresis or am I missing something?

[Yggdrasil]

The overall free energy change for unfolding is not different from that of refolding.  Rather, the hysteresis comes about because unfolding proceeds via an intermediate state that kinetically traps the native state and is not destabilized relative to the native state until much higher denaturant conditions than required for refolding to occur.  The authors have a nice diagram of the proposed energetics in a paper they published the following year (see Fig 1): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527903/

[urtula]

The overall free energy change for unfolding is not different from that of refolding.  Rather, the hysteresis comes about because unfolding proceeds via an intermediate state that kinetically traps the native state and is not destabilized relative to the native state until much higher denaturant conditions than required for refolding to occur.  The authors have a nice diagram of the proposed energetics in a paper they published the following year (see Fig 1): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527903/

If I understand you correct than the protein has to "reach" a intermediate state (the Niso) before it can unfold.

Simply put:

the energy to fold (same as refold I pressume?) is the same to unfold, but in order to unfold you first have to reach this intermediate state (meaning: first you need to invest some extra energy in the folded state to reach this intermediate state?

or?

A few extra questions:
first question: the horizontal bars in that figure (second part) they do represent energy levels right? THe higher the bar, the more energu?

A second question: this indermediate state (Niso) is this still an active fluorophore? I assume that in this state the protein is not working anymore as a fluorophore?

A last question: what are those red dots in that figure (second part figure 1)?

[Yggdrasil]

If I understand you correct than the protein has to "reach" a intermediate state (the Niso) before it can unfold.

Simply put:

the energy to fold (same as refold I pressume?) is the same to unfold, but in order to unfold you first have to reach this intermediate state (meaning: first you need to invest some extra energy in the folded state to reach this intermediate state?

Yes.  This energy corresponds to the energy required for a conformational change in the protein that isomerizes the chromophore.

A few extra questions:
first question: the horizontal bars in that figure (second part) they do represent energy levels right? THe higher the bar, the more energu?

Yes.

A second question: this indermediate state (Niso) is this still an active fluorophore? I assume that in this state the protein is not working anymore as a fluorophore?

Yes, in N_iso, the protein has folded to a near native state, but the fluorophore has not formed yet.

A last question: what are those red dots in that figure (second part figure 1)?

The dots represent the relative population of molecules in each different state.

[urtula]

If I understand you correct than the protein has to "reach" a intermediate state (the Niso) before it can unfold.

Simply put:

the energy to fold (same as refold I pressume?) is the same to unfold, but in order to unfold you first have to reach this intermediate state (meaning: first you need to invest some extra energy in the folded state to reach this intermediate state?

Yes.  This energy corresponds to the energy required for a conformational change in the protein that isomerizes the chromophore.

A few extra questions:
first question: the horizontal bars in that figure (second part) they do represent energy levels right? THe higher the bar, the more energu?

Yes.

A second question: this indermediate state (Niso) is this still an active fluorophore? I assume that in this state the protein is not working anymore as a fluorophore?

Yes, in N_iso, the protein has folded to a near native state, but the fluorophore has not formed yet.

A last question: what are those red dots in that figure (second part figure 1)?

The dots represent the relative population of molecules in each different state.

Ok thanks for the answers!

It is weird for me to see that in the "green area" the protein has to invest energy in order to reach this Niso form before it can unfold to the U form (that is lower in energy than the folded (Nnat) form!

So looking at this figure, its seems that the amount of protons play a big role in the formation of the either folded or unfolded form.
(protonation plays a big role).
Or is this not correct?

[Yggdrasil]

So looking at this figure, its seems that the amount of protons play a big role in the formation of the either folded or unfolded form.
(protonation plays a big role).
Or is this not correct?

Where does the diagram show protonation playing a big role?  As far as I know, protonation is not important for explaining the hysteresis.

[urtula]

So looking at this figure, its seems that the amount of protons play a big role in the formation of the either folded or unfolded form.
(protonation plays a big role).
Or is this not correct?

Where does the diagram show protonation playing a big role?  As far as I know, protonation is not important for explaining the hysteresis.

Well since they use guanidine-HCl plays a role, I was thinking the protons would matter? But its not than! Ok thanks.

[Yggdrasil]

The guanidine-HCl is a chemical that helps denature proteins by interfering with hydrogen bonding.  In that sense, protons are important in the process because hydrogen bonds are important in maintaining the overall folding of proteins.

[urtula]

The guanidine-HCl is a chemical that helps denature proteins by interfering with hydrogen bonding.  In that sense, protons are important in the process because hydrogen bonds are important in maintaining the overall folding of proteins.

Ok I see, it does play a role, but its not like I made a conclusion about it.
To me it looked it might play a role.
I still find it weird how the molecule behaves depending on the concentration (the intermediate form being either lower in engery or higher). Seems weird to me.

[TyPie]

I thought protons always mattered in long chain protein molecules.  I mean why wouldn't Van der Waal forces matter.  I can't see the paper but it sounds like you're explaining it right about the loop.