CD is sensitive to secondary structure context of biomacromolecules but not their tertiary or quaternary states. Tetrameric states shouldn't affect the CD spectra so long as the secondary structures remain the same.

Dear Wiktoria Brytan ,

As indicated by Yun-Tzai Lee circular dichroism gives indications about the secondary structure. If the transition of monomer to tetramer keeps the secondary structural elements unchanged, then no change in CD spectra will be observed.

However, whether this happens and whether CD spectroscopy can be of any use or not is highly unpredictable. In the literature you see all imaginable variations:

-There are differences in the various states, see for example Transthyretin Aggregation Pathway toward the Formation of Distinct Cytotoxic Oligomers

-There are differences in Tm, see for example Murugan S, Hung H-C (2012) Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme. PLoS ONE 7(12): e50143. https://doi.org/10.1371/journal.pone.0050143

-There are differences in folding depending on ionic strength and/or urea concentration, see for example Clark, A. M., Ponniah, K., Warden, M. S., Raitt, E. M., Smith, B. G., & Pascal, S. M. (2019). Tetramer formation by the caspase‐activated fragment of the Par‐4 tumor suppressor. The FEBS Journal, 286(20), 4060-4073

-Etc.

Back to your question I think it is hard to say whether your finding is relevant or not. The point is that the accuracy of the secondary structure deconvolution programs varies and when it comes to absolute values should be considered with great care. CD spectroscopy values are most valuable in getting indications (primarily helical, or unstructured etc.) and to monitor the relative changing effects of temperature, ionic strength etc. on the secondary structure level.

When it comes to what to expect and what can be predicted I would check:

-Use at least two different secondary structure prediction programs based ‘simply’ on the primary sequence (so insertion of the FASTA sequence). This would give you an idea about the monomer

-If possible the secondary structure determination of the tetramer which is based on the known structure (UniProt ) and/or predicted structure (Alphafold2) to get an idea about the secondary structure (content) of the tetramer, see for example Akdel, M., Pires, D.E.V., Pardo, E.P. et al. A structural biology community assessment of AlphaFold2 applications. Nat Struct Mol Biol 29, 1056–1067 (2022). https://doi.org/10.1038/s41594-022-00849-w

-Use at least two different CD deconvolution programs to check the circular dichroism spectra

-Do this for both the WT and truncated version of your protein

This might give you a good idea about what to expect (in terms of secondary structural changes). When it comes to the CD measurements, be sure you ‘understand’ your result, in terms of dependence of pH, temperature, ionic strength, are you in solution looking at the monomer or tetramer etc. In other words, be as sure as possible that when using a deconvolution program, you use a spectrum that represents the ‘true’ conformation of the protein (complex).

Sorry for the long story but the answer to your question is not that straightforward.

Best regards.