I would also like to know how you would differentiate between the point mutation results in the same amino acids?
Thanks for the help.
regards
Jitendra
Dear Jitendra,
Circular Dichroism (CD) probes the secondary structure of the protein while intrinsic fluorescence (fluorescence of tryptophan (W) and tyrosine(Y)) probes the tertiary structure. Intrinsic fluorescence can be used to probe (un)folding of only those proteins in which the hydrophobic environment of the amino acids (W and Y) changes during (un)folding studies. In cases where W and Y are exposed to solvent in the native folded state itself (present in loops, for instance), you can not use intrinsic fluorescence as a tool to probe (un)folding. CD on the other hand probes secondary structure which can help in carrying out protein folding/unfolding studies. Since during unfolding of a protein there will be atleast some secondary structural changes, CD helps to probe these changes.
In cases where W and Y are buried in the native state, intrinsic fluorescence can be used to probe (un)folding of proteins.
Best,
Jogender
Thanks Jogendra,
I would also like to know if you can answer in terms of spectra pattern?
Hi Jitendra,
In terms of fluorescence spectra when W and Y go into a buried hydrophobic core during folding from a polar environment in unfolded state, a blue shift in spectra is observed (shift of lamda maximum towards lower wavelengths). The degree of shift depends upon the amount of burial of the amino acids in the hydrophobic core. Hence the intensity of fluorescence at one particular wavelength will change with (un)folding of protein.
For better understanding of fluorescence I would recommend the book:
"Principles of Fluorescence Spectroscopy" by Lakowicz, Joseph R.
http://www.springer.com/chemistry/analytical+chemistry/book/978-0-387-31278-1
For CD you generally see a decrease in CD signal with unfolding of protein (from negative to towards zero). Since you get specific CD spectra pattern for different secondary structures (two minima for helix at 222 nm and 208 nm and one minimum for beta sheet at 216 nm), you probe the CD signal change at either 222 nm or 216 nm during (un)folding of the protein.
Cheers,
Jogender
Dear Yogendra your explanation is very lucid.
Great thanks for your kind help.
regards
Jitendra
Hello both! Just a note here in addition to your discusson that in case of intrinsically unstructured proteins e.g. milk caseins, the CD signals would be weak or without any specific pattern. In this case other methods such as intrinsic fluorescence could be more helpful to derive structral information about the proteins.
Hello Anant,
Its interesting to know this. Great thanks to bring this point up.'
Cheeres!
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