In addition to what has been said.

A tryptophan present on the surface of the protein is exposed to water / buffer (polar solvent) whereas a tryptophan buried deep site is in a hydrophobic environment.

Tryptophan fluorescence in proteins is sensitive to solvent polarity. As the polarity of solvent increases, fluorescence maxima red shifts and also quantum yield decreases.

Usually, as the tryptophan exposure to solvent increases, fluorescence quantum yield decreases and also fluorescence maxima red shifts.

On the other hand, there are also some proteins where tryptophan fluorescence gets quenched even though it is buried inside due to quenching by other residues.

That is the reason why protein fluorescence can increase or decrease due to changes in tertiary structure.

A good question.  In fact, intrinsic protein fluorescence (mainly from tryptophan residues) may actually increase or decrease when proteins are unfolding.  It all depends on the change in the local environment around the most strongly emitting residues.  One can also usually observe changes in the emission maxima as proteins unfold - usually the shift is to longer emission wavelengths.  Decreases in fluorescence polarization/anisotropy usually occur also. 

Sumit,

The two amino acids that fluoresce are tryptophan (emission max. ~ 280 nm) and tyrosine (emission maximum at ~276 nm).  Tryptophan with a much larger extinction coefficient and fluorescence quantum yield will be the most prominent emitter.  In general, the more buried these residues are within the protein the less fluorescence quenching they will experience.  Also, there will be less solvent induced fluorescence Stokes shift, which shifts the emission to redder wavelengths and over a broader spectral range.  If a change in tertiary structure increases the exposure of tryptophanyl or tyrosinyl residues to the solvent you can expect a decrease in fluorescence intensity.

I hope this helps.

Kind regards,

John

Dear Dr. David M Jameson and Dr. John E. Hansen,

Greetings!

Thank you for your  understanding answers. This really helped in clearing my concepts

With Best Regards

Sumit

The local environment around the fluorophore is determine the emission intensity and wavelength of a particular protein.  Total fluorescence of a protein is a mixture of the fluorescence from individual aromatic residues. During the tertiary structure stabilization several changes in the location and geometry of the individual fluorophore will take place. This will affect the total fluorescence positively of negatively. It will tell is there any structural changes happening around the flurophores of the protein. Change in the fluorescence of the protein have to be correlated with the results form other experiments like CD spectra to arrive at meaningful conclusion. 

In addition to what has been said.

A tryptophan present on the surface of the protein is exposed to water / buffer (polar solvent) whereas a tryptophan buried deep site is in a hydrophobic environment.

Tryptophan fluorescence in proteins is sensitive to solvent polarity. As the polarity of solvent increases, fluorescence maxima red shifts and also quantum yield decreases.

Usually, as the tryptophan exposure to solvent increases, fluorescence quantum yield decreases and also fluorescence maxima red shifts.

On the other hand, there are also some proteins where tryptophan fluorescence gets quenched even though it is buried inside due to quenching by other residues.

That is the reason why protein fluorescence can increase or decrease due to changes in tertiary structure.

Thank You Dr. V.N. Ravi Kishore

I completely agree with the above descriptions. When a protein unfolds the fluorescence intensity can increase or decrease depending upon the nature of micro-environment of fluorophores in the unfolded proteins. The increase or decrease in fluorescence intensity certainly reflect change in tertiary structure. However, variation in the fluorescence intensity alone may not be useful in assessing the changes in protein stability. 

Trp is indeed the most fluorescent amino acid followed by Tyr. Its fluorescent can either increase or decrase during folding. Work that I know that demonstrate this are attached. Trp fluorescence is often used as a mean of both determing stabiling and folding and unfolding rate (particularly for small protein displaying a two-state folding mechanism), but depending on the position of the Trp might vary particularly if the protein is complex and fold through multiple intermediate states. See also the review attached for a description of the standard experimental conditions for performing kinetic and equilibrium denaturation experiments.

Thank You Dr. François-Xavier Campbell-Valois. Your attached documents are really informative.

if fluorescence intensity is not showing a fixed pattern of either increase or decrease but it is randomly  decreasing or increasing then is it possible to calculate fraction folded/ unfolded from this type of data which is required for explaining the unfolding pattern and also for calculation of thermodynamic parameters of stability?

Hello Sarita Puri,

Can you please explain how I can explain the unfolding pattern using fluorescence data?

Hi Sarita,

Please view the following article under my ResearchGate site.   I hope it is helpful.

John

Article: A pH dependence study on the unfolding and refolding of apoazurin: comparison with Zn(II) azurin.  John E Hansen, Mary Kate McBrayer, Michael Robbins, and Yong Suh.  Cell Biochemistry and Biophysics 02/2002; 36(1):19-40.

Shashwati Atwe... Yes we can do unfolding and refolding studies by using intrinsic and extrinsic fluorescence. With various concentration of denaturant (urea, GdnHCl, Acid) etc. fluorescence  pattern of protein change.

John E. Hansen... thank you so much. this article is very helpful for unfolding and refolding studies for a protein. but sometime increase in fluorescence at high denaturant concentration is due to formation of intermediate in the unfolding and refolding pathway.

Hi everyone

I am having a similar problem regarding my protein denaturation studies.My protein is a tetramer and the intrinsic fluorescence intensity increases with respect to increasing GdnHCl concentration .Although there is a characteristic red shift in the spectrum.How can I analyse the data and is it possible to calculate the percentage of  denaturation from the fluorescence intensity.

HI Shreyasi,

If your protein is not monomeric than you must also take into account the subunit dissociation.  I am attaching a paper where we dealt with this type of problem to give you some ideas.

Good luck!

David Sir

One thing I would like to clarify here.I have read that for a simple two state unfolding mechanism,when we calculate the fraction of folded and free energy.Should they follow a straight line,when fitted?

hi everyone 

I am also facing similar type of problem, increase fluorescence intensity with increase urea concentration My protein is multi domain protein (3 sub-unit) so what will be the possible explanation for this.