I had used the intrinsic fluorescence of my protein to detect the change in fluorescence intensity with increasing urea concentration. I was astonished to find a huge increase in fluorescence intensity with increasing urea concentration? Can someone reason this phenomena of increased intensity with increased urea concentration?
Thank you in advance.
Here is an article about using protein intrinsic Trp fluorescence to monitor protein folding: Article Probing Protein Folding and Conformational Transitions with ...
This one takes about protein Trp fluorescence for FRETArticle Intrinsic Tryptophan Fluorescence in the Detection and Analy...
, both give nice introductions into the ways the environment of the protein affects the Trp fluorescence emission spectrum. For quenching, the emission energy of the fluorescent molecule has to overlap the absorbance spectrum of the acceptor to allow energy transfer from the fluorophore to the quencher. Trp is not significantly quenched by water, however, you can quench solvent accessible Trp residues with iodide as in this lab exercise:Article Protein tryptophan accessibility studied by fluorescence quenching
and thereby distinguish buried Trp residues from surface residues.In a native protein, Trp fluorescence quantum yield can be increased by energy transfer from Tyr residues, and quenched by disulfide bridges very close to the Trp. In addition, lambda(max) is very sensitive to the local environment, ranging from 308 nm (azurin) to 355 nm (e.g., glucagon) and roughly correlates with the degree of solvent exposure of the chromophore, that is, to whether it is in a hydrophobic or hydrophilic environment. Article Mechanisms of Tryptophan Fluorescence Shifts in Proteins
Of course, a shift in lambda max (the wavelength of the maximum of the fluorescence spectrum) to longer wavelengths upon unfolding means that the fluorescence intensity at wavelengths below the lambda max of the native protein decreases and at wavelengths above lambda max of the native protein increase upon unfolding, even if the area under the peak remains the same (no quenching, no resonance energy transfer). Quenching in the native structure will lead to an increase of the quantum yield upon unfolding, Tyr "feeding" Trp fluorescence in the native structure will lead to a decrease of the quantum yield upon unfolding.
Together, these and other, minor effects make it very difficult to predict how the fluorescence spectrum of a given protein with multiple Trp will change upon unfolding.
For example, the internal Trp in Antibody variable domains is almost completely quenched by the vicinity of the intradomain disulfide bond. Some VL domains have only this one Trp, these show a marked fluorescence increase upon unfolding. In scFv, the increase of the fluorescence quantum yield of the core Trp is masked by a decrease of the fluorescence quantum yield of the VL-VH interface Trp residues. As a result, some scFv show an increase, some a decrease of Trp fluoresence upon unfolding, some no change, but all show a red-shift of the fluorescence maximum.
Urea is a denaturant and destabilises proteins, causing them to unfold. As you have an increase in fluorescence intensity, the tryptophan and tyrosine residues, which are the natural fluorophores in proteins, are quenched in your folded protein. As the protein unfolds (with increased urea), the chemical environment of the tryptophan and/or tyrosine residues changes, removing the quenching and causing fluorescence to increase.
Additionally, small linear changes (increases or decreases) in fluorescence intensity can be observed as urea is titrated. These small changes are readily observed at low urea concentrations, where the protein structure is not substantially altered (fully folded), and at high concentrations, where the protein is fully unfolded (this small change in fluorescence is therefore not due to alterations in protein structure, but due to altered solvent conditions). Chapter 17 in this book (http://www.fersht.com/Structure_And_Mechanism_in_Protein_Science.pdf) explains it well, and discusses how to fit the changes in fluorescence to obtain the stability of your protein.
Thank you Dr.Annemarie Honegger and Dr. Michael D.Crabtree. I still do not understand why the fluorescence increases. Initially, the trp fluorescence is quenched by the residues and cys disulfide bonds in the folded protein which later upon addition of urea causes the protein to unfold and the intensity increases. Won't the fluorescence be quenched by water? Considering quenching be done by water upon unfolding, the intensity should decrease? Please correct me if I am wrong.
It is very hard to predict whether fluorescence will increase or decrease upon protein unfolding. It is certainly not a surprise for it to increase. I have seen it on numerous occasions and it just depends on the balance of quenching in the folded and denatured states. If the fluorescence is more quenched when folded then the fluorescence intensity will increase when it unfolds.
For a description, see Figure 1 (https://www.cell.com/biophysj/pdf/S0006-3495(94)80799-4.pdf).
Here is an article about using protein intrinsic Trp fluorescence to monitor protein folding: Article Probing Protein Folding and Conformational Transitions with ...
This one takes about protein Trp fluorescence for FRETArticle Intrinsic Tryptophan Fluorescence in the Detection and Analy...
, both give nice introductions into the ways the environment of the protein affects the Trp fluorescence emission spectrum. For quenching, the emission energy of the fluorescent molecule has to overlap the absorbance spectrum of the acceptor to allow energy transfer from the fluorophore to the quencher. Trp is not significantly quenched by water, however, you can quench solvent accessible Trp residues with iodide as in this lab exercise:Article Protein tryptophan accessibility studied by fluorescence quenching
and thereby distinguish buried Trp residues from surface residues.In a native protein, Trp fluorescence quantum yield can be increased by energy transfer from Tyr residues, and quenched by disulfide bridges very close to the Trp. In addition, lambda(max) is very sensitive to the local environment, ranging from 308 nm (azurin) to 355 nm (e.g., glucagon) and roughly correlates with the degree of solvent exposure of the chromophore, that is, to whether it is in a hydrophobic or hydrophilic environment. Article Mechanisms of Tryptophan Fluorescence Shifts in Proteins
Of course, a shift in lambda max (the wavelength of the maximum of the fluorescence spectrum) to longer wavelengths upon unfolding means that the fluorescence intensity at wavelengths below the lambda max of the native protein decreases and at wavelengths above lambda max of the native protein increase upon unfolding, even if the area under the peak remains the same (no quenching, no resonance energy transfer). Quenching in the native structure will lead to an increase of the quantum yield upon unfolding, Tyr "feeding" Trp fluorescence in the native structure will lead to a decrease of the quantum yield upon unfolding.
Together, these and other, minor effects make it very difficult to predict how the fluorescence spectrum of a given protein with multiple Trp will change upon unfolding.
Several years back, we have seen similar effects, while following Urea denaturation curve of purified recombinant mycobacterial cAMP receptor protein. As nicely explained above, the effect is may be due to the exposure of buried Tryptophan residues to the solvent. Here is the link of the published paper on the same story : Article Novel biochemical properties of a CRP/FNR family transcripti...
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