Hello Sorin,
1. The cost of getting NMR data on the IDPs you propose depends upon the
expression levels of the protein/s as you will need to label with 15N and 13C -
and depending upon your overall yields per liter of E.coli culture, this can
add up. In addition you will need to run triple resonance experiments - so you
should look into the hourly charge to access the NMR spectrometers where you
are located. Moreover, you need to account for time required for optimization
of solution conditions to collect the NMR data as the sample needs to be
homogenous (as in no aggregation) at millimolar or hundreds of micromolar
concentration. As Ethan Merritt suggested, it would be a good idea to use
SAXS first as it requires very little sample, no isotope labeling, and you can
try to narrow down the solution conditions that would be best suited for NMR.
The Kratky plots, Rg values under different solution conditions can give very
useful information about conformational states and ensembles populated by IDPs.
However, although NMR tends to be more expensive than other techniques but is
perfect for IDPs as you point out you can get residue specific information. A
combined NMR/SAXS approach has proven to be very useful to validate
computational models.
2. In general, CROs are much more expensive particularly for generating
isotopically labeled samples - it is cost-prohibitive for academic labs.
Genscript is one CRO that will express proteins, but I am not sure if they will
make isotopically labeled proteins for NMR.
3. The amount of protein needed depends upon the size of the molecule. You
will need at least 2-3 samples that are differentially labeled with 15N, 13C
(also since you want data on the free and bound forms of the complex) at 0.5 -
1 mM depending upon the size of the molecule which relates to the complexity of
the NMR spectrum due to number of resonances and the relaxation times. The
total volume required for each sample is between 280 ul - 600 ul, depending
upon which type of instrumentation and NMR probes you have access to.
Hope this helps!
Best regards,Roopa
On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga
<sor.dr...@gmail.com> wrote:
Hello everyone,
I do realize that this is not a NMR focused group, but I do hope that there are
a few spectroscopists lurking around that could possibly answer a few questions
(I am more of a modeler/computationalist):
The problem: I have two intrinsically disordered proteins that are known to
interact (let's call them 1 and 2). I would like to get structural information
(a conformational ensemble) for 1 and for the "complex" (1+2). Further down the
line (depending on whether this is possible) I would also like to evaluate
potential small molecule inhibitors for the said complex. Both 1 and 2 are <200
aminoacids long.
The questions:
1. Could the cost of determining the "structure" for 1 and 1+2 be estimated? To
be more precise, I am looking for a ball-park figure on how much a NMR
measurement would cost in this case. 2. Could anyone recommend a good group/CRO
that could provide such a service and not have an astronomical cost?3. Any
other suggestions/thoughts that you think might be worth mentioning (minimum
quantity of protein necessary, purity, type of NMR etc)
Many thanks for your help and time!
Cheers!
Sorin
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