Question

Challange: Folding and thermodynamics

Answer 1

BRIC is a protein that exists as a dimer. Equilibrium guanidinium chloride (GdmCl) unfolding probed by circular dichroism (CD) spectroscopy at 298 K showed two transitions with the fitted parameters in Table A.
Moreover, denaturation was measured at several different protein concentrations from 1.5 μM to 18.8 μM (Fig. 4. A, B.) The far‐UV CD spectrum of BRIC was recorded at different concentration of GdmCl from 0 (black line) to 7.5 M GdmCl (grey line top), Fig. 4.C.

Figures attached.

Table A
\(\begin{array}{|c|}
\hline
\ Transition& \ C_m (M)&\ m(kJ/(mol*M) \\
\hline
1&1.97& 3.1\\
\hline
2&5.32&3.6\\
\hline
\end{array}\)

Describe the unfolding reaction of BRIC using the given data.

Answer 2

@Kainui

Answer 3

@Mommy :O

Answer 4

Also info can be found here :P
https://en.wikipedia.org/wiki/Equilibrium_unfolding

Answer 5

And CD here:
http://www.proteinchemist.com/cd/cd7.gif
https://en.wikipedia.org/wiki/Circular_dichroism

Answer 6

(reaction kinetics I don't feel the needs to tell as this is chemistry!!!!)

Answer 7

Questions that could help solve the problem:
* What is the dominating secondary structure of BRIC?
* What is the conformational stability of BRIC?

Answer 8

Ok I think I'm starting to understand C of the Far-UV CD spectra. Since it's a measure of the ellipticity, which is really just the difference in amount of chiral light absorbed we can see as we increase the concentration of GdmCl that this deviation decreases so BRIC must be untwisting.

It appears that they've done two measurements at 0 and then again at 1.5 before the first transition. After the transition they seem to have done two as well to mark the end and then the start of the next transition where it untwists yet again. and then they measure it at completion.

I'm not really sure what this particular wavelength means, if anything, and it seems as though it might be untwisting a little bit more in the first transition than it does in the second, but it's roughly the same I guess.

My hope was that part of the time it would be untwisting and the other transition would be it cracking the dimer into two pieces. I'm not sure what this \([GdmCl]_{50\%}\) thing is, but I'm gonna look into it in a bit after I grab a snack lol.

Answer 9

Keep the last though and build on with that. The [GdmCl]50 can be considered the concentration which 50% of the population is denatured.... could it maybe be related to some kind of kinetics? ;)

Answer 10

In other words: does both unimolecular and bimolecular reaction depend on the concentration?

Answer 11

It's 5am here, I think I'm gonna go to sleep. I'll be back!

Answer 12

haha, Sleep tight! :)

Answer 13

ANSWER:
The first transition is independent of the concentration of BRIC suggesting it to be a unimolecular reaction, while the second transition is depending on the concentration suggesting a bimolecular reaction. As BRIC is a dimer, the unfolding of the dimer happens in the second transition. After the first transition the far-UV CD spectrum show the intermediate still have secondary structure and hence not unfolded.

It is therefore suggested the first transition unfolds the dimer into a dimeric unfolded state which is also in accordance with the small m-value showing the newly exposure of surface is small. While in the second transition the unfolded dimer dissociates and completely unfolds, as shown in accordance with the CD spectrum and the larger m-value together with the concentration dependency.

@Kainui

Answer 14

\(\sf N_{2} \rightarrow D_2 \rightarrow 2 D\)