Trp fluorescence tells about the environment around the trp residue(s) and CD gives secondary structural content. So, delG from two experiments might be different.

Obviously, sometimes the values might be similar provided the transition is two state and reversible.

What Nilimesh says is right . At high concentration of urea the protein will be fully denatured. At that condition both the methods will be irrelevant. I feel DSC is the the better method to calculate dG.

Thanks Nilimesh Das and Agnishwar Girigoswami Sir. In my case protein unfolding curve is well fitted into two state model (as shown in picture). So the difference in Delta G might be due to unfolding of tertiary structure by Trp (6 Trp) fluorescence. As in case of CD fraction unfolding curve, the pre-transition region is no prominent if compared to fluorescence unfolding curve. Does this also reflect some kind of intermediate, which could not be track down by Urea unfolding?

Don't you feel that these two principles are different. In this case intrinsic fluorescence is just representing the change in the micro environment around the Trp. CD is an absorption phenomenon.

There could be three reasons: First, unfolding of tertiary structure (as shown by trp emission) and unfolding of secondary structure (as shown by CD) might be different. Secondly, as you have said the pre-transition region is missing. So, the assignment of the Initial/native/folded state might be wrong. Thirdly, the transition involving CD has not been fitted well. It seemed to me a three state transition. At low urea concentration, a deep and then increase. The trp fluorescence can not capture the presence of intermediate state around 2 M of urea; however your CD data can capture this intermediate state. This is also very much usual that with some technique you find such intermediate and with some other technique, you do not as different experiment track different information. At lower urea concentration, protein structure becomes more compact is also very

usual.

I have serious concerns with interpretation of fluorescence changes as changes in protein tertiary structure. Did you try to use near-UV CD, which is more directly related to protein tertiary structure? What parameter did you use in your analysis of protein unfolding by fluorescence - intensity at fixed wavelength, integral intensity or position of maximal fluorescence? Do you have any information on changes in protein compaction during urea-induce unfolding?

As Vladimir has pointed out, the question posed lacks proper background information to answer it correctly. Please provide more data from other areas to help you with the answer.

Dear Professor Vladimir N Uversky and Wieslaw Swietnicki i have taken 2.5 uM helicase protein for urea denaturation experiment by fluorescence measurements. The measurements were taken by exciting at 295 nm and spectra were recorded between 300 to 50 nm. The protein is having molecular wight of aprox 53.6 Kda, containing three domains and six tryptophan residues. The urea exchange titration experiment has shown that at 0M urea the max emission was found at 325nm, with increasing urea concentration the emission max shift towards red (as trp exposed to polar environment) with max emission of 341nm at 8M urea (as shown in figure). Finally the fraction unfolding curve was checked at 315 nm wavelength. This wavelength was chosen because the spectra of unfolded and folded conformation differs significantly.

I have not checked the near UV spectra. i will check that too.

Dear Deepak,

Let me explain it plainly. Trp residues are in different environments and each has a different contribution. As a rule, the more hydrophobic the bigger change in properties upon exposure to the solvent. What you measure is a combined contribution of Trp residues to the observed spectrum which may have nothing to do with the overall structure.

CD measurements give you changes in the overall secondary structure. Depending on the conditions and the signal, you may monitor it at 222 nm (a-helical content) or at 195 nm which has contributions from many secondary structures. The change you measure is an overall contribution from secondary structures.

Tertiary structure is something different than the secondary structure. You may measure changes in it by SEC, radiation scattering and many other techniques.

Your data does not provide any hint you have taken into account the phenomena just described. Is this clear to you now?

Thanks prof Wieslaw Swietnicki . I got it. In other words , studies have also shown that the non-coincidence of equilibrium unfolding plots elucidated from different techniques prove the presence of folding intermediates.

Dear Deepak,

Thanks for sharing these data. The fact that transition curves measured by different techniques do not coincide is a strong indication of the presence of a partially folded intermediate. However, to get into nature of this intermediate, you need to conduct more experiments.

I also would like to see far-UV CD spectra you measured.

The best low-resolution techniques for the analysis of changes in tertiary structure is near UV CD. SEC, viscometry, and light scattering will allow you to evaluate changes in protein compaction (which is another important structural feature).

Shifts in maximal Trp fluorescence which reflects the degree of solvent exposure of Trp residues is a better reflection of the overall compaction of a protein molecule than protein tertiary structure. If you will repeat your fluorescence measurements in the presence of 0.1 M acrylamide, you will measure urea-induced changes in compaction more precisely.

You can re-analyze your data using phase diagram method described in PMID: 15253430.

Hope this helps.

If you need further help, please let me know.

Vladimir

Thank you very much Professor. Your suggestions are very logical. I will perform the experiment with acrylamide and near UV CD.

OK. Please keep me posted about developments in this project. Also please contact me if any other help will be needed

Sure Professor, i will be keep discussing and posting you about this data. Thank you very much.

Dear Deepak,

Please do. When you have more data from other systems, you may start drawing conclusions.

Observation of discrepancies between different approaches(UV, Fl, CD) may indicate the presence of an intermediate. It may also suggest that the techniques measure different properties and your observations may reflect changes specific for a particular approach.

A true folding intermediate is hard to isolate as it is most unstable. However, showing that under certain conditions you obtain species which aggregate may simply hint at the presence of such species if there is another set of experiments supporting it.

The kinetics of unfolding which may give you a hint if there is an intermediate. It takes time and requires a lot of protein not to mention a stop-flow system coupled to something (CD, FL, UV).

Wish you good luck with your experiments,

W

Thank you very much Wieslaw Swietnicki

Dear Deepak,

Please do. When you have more data from other systems, you may start drawing conclusions.

Observation of discrepancies between different approaches(UV, Fl, CD) may indicate the presence of an intermediate. It may also suggest that the techniques measure different properties and your observations may reflect changes specific for a particular approach.

A true folding intermediate is hard to isolate as it is mostly unstable. However, showing that under certain conditions you obtain species which aggregate may simply hint at the presence of such species if there is another set of experiments supporting it.

Try kinetics of unfolding which may give you a hint if there is an intermediate. It takes time and requires a lot of protein not to mention a stop-flow system coupled to something (CD, FL, UV).

Wish you good luck with your experiments,

W

Your curves show that the protein unfolds with intermediate state, since the CD and fluo unfolding curve is not overlapping. Though it is appearing biphasic. To confirm this, u should perform CD and fluorescence studies at different protein concentrations (may be in log ratio, as 1, 10, 100 concentrations). The curves now obtained for either CD/fluo will not overlap i.e. the CD curves will have different Cm values. This will confirm the folding to be non-co-operative. Additional experiments can be done, u can discuss with me if needed or even rajanish may help u out.