Answered: Help!!Why does a deuterated protein behave even more poorly than the protonated one?
A 20kDa protein, about half of the total signals can be observed in CBCA(CO)NH experiment when protonated with sample concentration of 1mM. However, after deuterated, hardly any signals can be observed with the same concentration in this experiment. We use the same pulse sequence except adding deuterium decoupling in the deuterated one. I don't know why? Help!!
Was some effort made to allow amide exchange to occur, ie unfolding and refolding in H2O, before you ran your spectra? If not you may not have enough amide protons there for HN experiments to work. Do you get a good N15 HSQC with the same number of transients that worked for the protonated sample?
You might also try to compare a TROSY-HSQC with a regular HSQC to see if that helps the sensitivity although for a globular 20kDa protein in a spectrometer field lower than 700 MHz the sensitivity gain will probably be marginal.
The CBCA(CO)NH experiment depends on an initial 1H-13C transfer if your protein is deuterated you have no 1H's to excite on the carbons so you will get no signal, when you have a deuterated protein you have to start on the HN groups so you run a HN(CO)CACB instead.
Was some effort made to allow amide exchange to occur, ie unfolding and refolding in H2O, before you ran your spectra? If not you may not have enough amide protons there for HN experiments to work. Do you get a good N15 HSQC with the same number of transients that worked for the protonated sample?
You might also try to compare a TROSY-HSQC with a regular HSQC to see if that helps the sensitivity although for a globular 20kDa protein in a spectrometer field lower than 700 MHz the sensitivity gain will probably be marginal.
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[Question from NMRWiki Q&A forum] caluclation of duterium percentage of randomly deuterated protein
caluclation of duterium percentage of randomly deuterated protein
Dear NMR WIKIERS ,
IS THEIR ANY NMR EXPERMENT ON 700 VNMR TO KNOW THE duterium percentage of randomly deuterated protein WHICH PREPARED USING 90 PERCENT D20 AND 10 PERCENT H2O ?
Regards
SRI
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If a protein really does not behave, try this one
Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2 alpha.
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Abstract:
Linear Mode
