[Demelerlab] AUC results

[Borries Demeler]

Hello Yogesh and Jyoti,
Kate and Sophia performed your AUC experiment and analyzed the results with
all of our tools in our toolbox.
I am happy to report that the results are completely unambiguous, despite
some issues along the way, such as the thawing of the sample during
shipping. Despite the thawing event, the protein remained perfectly stable
and did not degrade or aggregate. So the samples you sent were in really
good shape even after the thaw.

Another problem we came across was that the buffers you sent appeared
contaminated with nucleic acids to varying degrees. This was apparent from
their absorbance spectrum we measured in our spectrophotometer, and can be
seen readily from the absorbance spectrum, and the peak at 260 nm:


[image: image.png]

Our spec was blanked with water and then the red line is our PBS buffer.
All other buffers, incl. your PBS, had some degree of absorbance at 260 nm,
but not our PBS or cuvette. So the 260 absorbance clearly comes from your
preparation. You should find the source of this contamination. Fortunately,
the amount of this absorbance was less than 0.25 OD for the worst
contamination, and did not interfere with the measurements we made.

We measured a low concentration sample measured at 239 nm, and a high
concentration sample (~ 1.3 mg/ml) at 295 nm. In both wavelengths there was
minimal contribution from the contaminant, but we were still able to see a
smaller species sedimenting at about 10 kDa, especially in the low
concentration samples. We did not investigate these contaminants further,
and I don't think the contamination has any impact on our
results, nevertheless, you should be aware of this.

Now to the results: All samples, regardless of concentration, unambiguously
showed identical results with only a monomeric sample present. The molar
mass was spot on for the known molar mass of the monomeric NUDT3, as you
can see from our overlaid 2DSA-Monte Carlo results:

[image: image.png]

Kate also did a global genetic algorithm Monte Carlo (GA-MC) analysis over
all samples and determined molar mass, frictional ratio, and s and D
values. Here is a pseudo-3D plot that indicates the solutes found from a
globally combined GA-MC analysis for all samples:


As you can see, all samples return values very close to each other, with a
moderate anisotropy of ~ 1.4.
The statistics of this fit for the boxed solutes are shown below:

[image: image.png]

*Solute 1:*

*Molecular weight: 1.9880e+04 (1.6212e+04, 2.3548e+04)*

*Sedimentation coefficient: 1.8990e-13 (1.6992e-13, 2.0988e-13)*

*Diffusion coefficient: 8.5097e-07 (7.2433e-07, 9.7760e-07)*

*Frictional ratio: 1.4111e+00 (1.2704e+00, 1.5518e+00)*

*Partial specific volume: 7.2730e-01 (**constant**)*

*Partial concentration: 5.9745e-01*

As you can see, the molar mass is in excellent agreement with the monomeric
mass, and no mass action effects are seen when comparing low and high
concentration samples, indicating that there is no difference in
oligomerization state in any of these samples. The molar mass plot shows no
indication of any higher oligomeric species:

[image: image.png]

Furthermore, and overlay of the van Holde - Weischet G(s) plots also
indicate perfect homogeneity, reflective of a monomeric species:

[image: image.png]

All of these plots are uploaded to your LIMS account. Kate will write a
report on this experiment and share it with you.
Sophia will schedule a time for a zoom call to discuss these results
further and decide what other experiment should be performed to explain the
difference in activity of your proteins.

Best wishes, -Borries
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