Replies

• Michael Durney

  01 September 2007 | 02:15

  As a basic scientist my knowledge of the human body is sometimes shockingly bad. So I looked up plasma in the dashboard dictionary on my Mac and here is what it said:

  The colorless, fluid part of blood, lymph, or milk, in which corpuscles or fat globules are suspended……

  I presume that this is the plamsa you refer to. As far as I understand contrast reagents are often used in MRI to examine organs but these are simple molecules. Labeling proteins for in cell NMR is really a new field and is still being explored using model systems such as oocytes. Maybe the results would be interesting but I’m not sure how the experiment could be designed to give some indication of what happens in the plasma in the body.

  Why the interest in plasma? Does something unusual happen to proteins there?

• Bronwen Dekker

  01 September 2007 | 08:59

  I suppose the question interests me, because plasma is a biological matrix that is liquid and easy to obtain, so the experiment seems like it would be relatively ‘easy’ to perform (in comparison with the experiments in cells).

  Perhaps it is only a really good matrix for proteins which perform their function in the blood, but I can’t help thinking that it would be interesting even for other other proteins, just to get a feeling for the differences in structure that may occur in solutions that contain complex mixtures of proteins, sugars etc.

• Nick Anthis

  26 September 2007 | 23:24

  One of my colleagues just did an experiment adding tissue lysates to a protein sample, and it seemed to work fine, although he didn’t notice any changes in the spectrum of the protein he was observing (he was hoping to identify a novel ligand).

• Bronwen Dekker

  27 September 2007 | 08:28

  Gosh! It is interesting that there weren’t any changes in the spectrum… perhaps that it why the experiment hasn’t made it into the literature?!

  Before your colleague did the experiment, had he read or heard of anyone who HAD seen evidence of binding to known or new ligands by doing a similar experiment?

  I suppose that the protein might be in quite a high concentration in comparison to the potential ligands… In the days when I did nmr, it was actually really difficult to see a minor component (in this case the protein-ligand molecule) unless its concentration was more than about 10 % of that of the main component. Things have progressed a lot since then, no doubt…

  I wonder if it is possible to concentrate the lysate without turning it into brown goo. :)

• Philipp Selenko

  05 October 2007 | 01:34

  you are right. the typical concentrations of NMR samples are in the umol to mmol range. in order to ‘detect’ interactions with endogenous components, either in plasma, or in any other unlabeled complex mixture for that matter, ligands have to be present at roughly equimolar concentrations. (keep in mind that you might have interactions at substochiometric ratios, however you will not pick them up because most of your NMR signal arises from the free biomolecule. the there is the affinity and the resulting timescale problem for NMR, but that’s another issue). physiological concentrations of most biomolecules are simply not in this range (neither in biological fluids nor inside whole cells). Hence, picking up endogenous interactions will be extremely difficult with current techniques (DNP might help at some point in the future).

  another caviat is that most ‘intact’ biomolecules are larger than the current size limit for NMR. remember that in biological fluids, macromolecules are not present as small protein entities, as in vitro NMR likes to believe, but as full length proteins which are on average >100kD. most binding events will hence result in NMR signal cancelation and provide no specific information whatsoever. not exactly encouraging is it?

  you have to think differently if you want to come up with useful in-cell NMR applications. how about biological activities that ‘modify’ your in-cell NMR sample? I think enzymatic reactions (in the broadest sense) are the way to go. by definition, enzymes will catalyze reactions irrespective of substrate concentrations. and some of them are most amenable for NMR analyses.

• Alex Hansen

  18 April 2008 | 14:05

  I think the prospect of in-cell NMR is very interesting and feasible. Someone in our group is working on an in-cell RNA NMR project and has done several steps in that path: used free, PEG, then cell extract, and finally in-cell itself. Interestingly, the only differences that show up appear to be the same varying salt concentrations. That, and the cells chewed up the RNA in about 6 hours :P Not much time for an NMR experiment!