A difficult question to answer. However, you can use a standard biochem hypothesis - A folded protein with deep interior cores will not easily exchange (may not exchange at all). When it is unfolded (either in vitro by strong denaturants or in vivo during folding of the nascent polypeptide expressed), protons should exchange readily. Hope it helps.

Dear Subhash

first of all a question: how was yor protein purified?

Did you usedbuffers in 100% D20?

Regaridng the expression to my knowledge using m9 media with 100% of D20 you can reach high labelling % , however to reach % >90% you need also to feed the bacteria with deuterated glucose in the M9 media instead standrds glucose.

eg. https://www.protein-nmr.org.uk/general/isotopic-labelling/15n-13c-2h/

moreover is it important once you produce the deuterated protein. mantain it in buffer with deuterated water otherwise you can have the exchange of the exposed OH and NH hydrogens (but not the Ch) and lost their deuteration.

This phenomena is exploited by the H/D exchange mass spectorrometry and NMR approaches to map the protein surfaces or the regions involved in protein protein interactiob by y detecting the hydrogens that are exchangable in the single proteins but not in the protein complexg

https://blog.acdlabs.com/elucidation/2008/03/how-do-i-know-w.html

good luck

Manuele

We used this approach to study the complex where the protein expressed in the deuterated water and glucose. 100% deuteration is practically impossible unless you started with unfolded protein and then refold it in the deuterium. Still, it is not 100% because you always have some humidity around. Protons can easily exchange.

Coming toward the end of your question: exchangeable protons are the main-chain peptide group NH and the side-chain protons bound to N, O and S atoms of polar groups

Article The H/D-exchange Kinetics of the Escherichia coli Co-chapero...