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  #1  
Unread 11-13-2006, 01:35 PM
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Default 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!!
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Best Answer - Posted by mrevingt
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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Unread 11-13-2006, 04:27 PM
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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.
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Unread 11-14-2006, 01:35 AM
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Thank you for your enlightening answer, but we also got poor signal in the HNCA experiment, I don't know why.
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Unread 11-15-2006, 04:41 PM
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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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