[Demelerlab] AUC results

Sun Jan 7 20:11:34 MST 2024

Dear Dr. Demeler,
Thanks to you, Kate, and Sophia for performing all the experiments, and analyzing the data. This is good to know that protein is stable, and I have no idea how nucleic acids were there in the buffers. I will use nuclease free water next time for such experiments. But what I can see from the results that it is a stable monomer at all the buffer, and pH conditions provided.
Next, I would like to try low salt conditions and some of divalent cations in the buffer.
It would be great to discuss more on zoom meeting.

Thank you for your time!
With best regards,
Jyoti

Get Outlook for iOS<https://aka.ms/o0ukef>
________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Sunday, January 7, 2024 2:43:14 PM
To: Gupta, Yogesh K <GuptaY at uthscsa.edu>; Baranwal, Jyoti <baranwal at uthscsa.edu>
Cc: demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: AUC results

*EXTERNAL EMAIL*

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.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.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.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.png]

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

[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

CAUTION: This message originated outside of UT Health San Antonio. Please exercise caution when clicking on links or opening attachments.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0001.html>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 97683 bytes
Desc: image.png
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0005.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 168342 bytes
Desc: image.png
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0006.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 97244 bytes
Desc: image.png
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0007.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 94850 bytes
Desc: image.png
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0008.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 248796 bytes
Desc: image.png
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20240108/024b59d4/attachment-0009.png>