Hi all!
I would like to study the interaction between free L-Cysteine and disulfide-bonds in proteins (e.g. whether the disulfide-bond between proteins gets "broken" and a new disulfide-bond between the free available cysteine and the reduced thiol group of the cysteine in the protein can form.
In fact, I would like to know whether free available cysteine can inhibit/ promote the crystallisation of proteins by interacting with the disulfide-bond. However, I am aware that cysteine reacts with cysteine to cystine (by forming a disulfide-bond under condensation with the dissolved oxygen in solution), which is obviously detrimental to the formation of the disulfide-bond between a free cysteine and the thiol group of a cysteine in the protein. Additionally the solubility of cystine is significantly lower than of cysteine and hence cystine will precipitate out from solution. Hence I bubble N2 through the solution prior to crystallisation to remove all dissolved O2 and replace it with N2.
However, since using a hanging drop vapour diffusion set-up, I am not sure how I can remove all the O2 within the plate prior to setting up the experiments? And whether cysteine can actually interact with a disulfide – bond withion a protein? Any idea of how I can tune my set up to study the interaction of cysteine with the disulfide-bonds in proteins in a O2 free environment?
Many thanks! Frederik
That's outside my knowledge, but you could get a heavy metal bridge between the two
Dear Frederik Link thank you for your interesting technical question. In this context please have a look at the following potentially useful article:
Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR
https://www.frontiersin.org/articles/10.3389/fchem.2020.00280/full
Dear Frank T. Edelmann that you very much for this useful publication.