Hello,
I intend to perform thermal unfolding of my proteins by CD, to determine their melting temperatures.
I initially scanned the proteins over a wavelength range of 250 -190 nm at 25 °C (in 20 mM Phosphate bufffer pH 7.4) to determine their structural content. The results confirm that the proteins are made up of alpha-helices (which is what i expected), with two negative bands at ~208 nm and ~221 nm, and one positive band at ~ 192 nm . See attached image!
Next, I will like to thermally unfold the proteins at a specific wavelength. Given the results attached, could anyone please suggest what wavelength would be idle to monitor the thermal denaturation of the proteins and why?
Best
Hi
222nm is recommended
Thermal scan rate between 0.5 upto 1 c/min is good.
Also linear voltage dynode condition is needed.
Please dont hesitate for contact, if mor help and explanation is needed.
Yours truly.
If you are interested in monitoring the denaturation of the alpha-helical secondary structure, then 222 or 208 nm would be good choices, since they have the largest signal in the far-UV (the 190 nm peak can be difficult to work with because of interference from the buffer). If you are interested in monitoring the unfolding of the tertiary structure but not necessarily the secondary structure, then I would suggest monitoring changes in the near-UV at 280-290 nm.
Asadollah Asadi and Adam B Shapiro thank you very much for your answers.
Do you think it makes a huge difference if the proteins are melted at a rate of 2°C/min or 1°C/min? I have over 10 mutants to melt in at least triplicate measurements each (from 25 - 95 °C). For the sake of time, I would like to speed up the temperature ramp as much as possible, without decreasing the quality of the results.
The influence of scanning rate depends on whether your protein unfolds reversibly or irreversibly.
For a reversibly unfolding protein, the scanning rate will not affect the stability parameters (mainly, unfolding temperature and enthalpy), but for an irreversibly unfolding protein, the scanning rate will strongly modulate the stability parameters.
In the first case you may estimate the equilibrium unfolding parameters (Tm, ΔH(Tm), ΔCp) from which the stability profile (ΔG vs. T) can be outlined.
In the second case you may estimate the kinetic unfolding parameters (k, Ea) from which the half-life (as in an accelerated aging test) can be estimated.
Therefore, if you have a panel of proteins, they may behave slightly different regarding (ir)reversibility of unfolding.
Dear Cynthia Bessem Nkemeto ,
The earlier mentioned 222 nm find its origin in the following source: The ellipticity at 222 nm has been used as a rough measure for the relative helicity, for which ellipticity222 = 36300 deg.cm2/dmol was taken as 100% alpha-helix (Hodges et al., 1988).
Hodges, R. S., Semchuk, P. D., Taneja, A. K., Kay, C. M., Parker, J. M., & Mant, C. T. (1988). Protein design using model synthetic peptides. Peptide Research, 1(1), 19-30.
You can find the paper here: Article Protein design using model synthetic peptides
and in Figure 4 (they actually used 220 nm) examples of thermal melting profiles).Best regards.
See for an example where this "222 nm" approach is used and compared with another attempt to estimate the secondary structural elements:
Article Anionic phospholipids are essential for α-helix formation of...