While calculating the percentage protein unfolding through equilibrium unfolding by intrinsic tryptophan fluorescence method, people generally plot intensity at single wavelength (e. g. 340nm) with concentration of denaturant (urea or guanidine). In some reports however, people calculated the ratio of fluorescence intensity at 2 different wavelength (360nm and 320 nm) and plotted the ratio against denaturant concentration (Melissa S. Kosinski-Collins, Shannon L. Flaugh, and Jonathan King; protein sc.). I performed equilibrium unfolding experiment on one of thermostable homodimeric protein (each subunit consist of 2 domains), and calculation of del G by 2 above mentioned methods yield different results. Also, while denaturation plot at single wavelength (340nm) displayed 3 state protein folding, denaturation plot at ratio of 2 wavelengths ( 353/333) displayed 2 state folding behavior. Interestingly, the equilibrium denaturation curve by plotting far UV CD signal at 222 nm coincide nicely with (353/333) curve. I am in a confusion about the selection of the appropriate method in case of my protein. While complex 3 state folding behavior is expected for a dimeric 2 domain protein, the rather unambiguous far UV CD method yields 2 state folding behavior.
If the transition occurs between two states (folded and unfolded) it's more correct to use fluorescence intensity at a fixed wavelength or fluorescence quantum yield - these parameters are linely related with the extend of unfolding. The ratio of fluorescence intensities at two different wavelengths is not related linely with the degree of unfolding in general case. If the unfolding passes through an intermediate state it's better to consider two separate transitions.
In fact, since wavelength shifts may accompany unfolding, I think its best to measure the intensity of the entire emission spectrum (corrected if possible). Furthermore, if the polarization changes (which is likely) then it is better to excite the sample with parallel (vertical) polarized light and observe emission through parallel and also perpendicular emission polarizers and then use the function I(para) plus 2xI(perp) which represents the total emission intensity corrected for any changes in polarization. This procedure was outlined in the paper Lasagna et al "Apohorseradish Peroxidase Unfolding and Refolding: Intrinsic Tryptophan Fluorescence Studies: Biophysical Journal (1999) 76:443.
Permyakov, E.A. Luminescent Spectroscopy of Proteins. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1993
Dushyant,
Please check out the following reference, which can be found on my ResearchGate webpage.
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, Yong Suh
Cell Biochemistry and Biophysics 02/2002; 36(1):19-40.
Cheers,
John
Dushyant,
Please check out the following reference, which can be found on my ResearchGate webpage.
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, Yong Suh
Cell Biochemistry and Biophysics 02/2002; 36(1):19-40.
Cheers,
John
Article A pH Dependence Study on the Unfolding and Refolding of Apoa...
You can try and calculate average wavelength. Please refer to the following paper for calculations
Moon and Fleming, Methods Enzymol., 2011, 492, 189-211
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