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<p class="MsoNormal"><span style="font-size:12.0pt">Thank you very much Bories, Kate, and Sophia!<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt">Jyoti and I will discuss the results and get back to you.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt">Yogesh<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal">Yogesh Gupta, Ph.D.<br>
Associate Professor<br>
University of Texas Health Science Center at San Antonio<o:p></o:p></p>
<p class="MsoNormal">Department of Biochemistry and Structural Biology<o:p></o:p></p>
<p class="MsoNormal">Greehey Children's Cancer Research Institute<br>
8403 Floyd Curl Drive, MC7784<o:p></o:p></p>
<p class="MsoNormal">San Antonio, TX, 78229, USA<br>
Phone: 210-562-9064<br>
<a href="http://ccri.uthscsa.edu/YGupta.html"><span style="color:#0563C1">Gupta Lab</span></a><span style="font-size:12.0pt"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><b><span style="font-size:12.0pt;font-family:"Aptos",sans-serif;color:black">From:
</span></b><span style="font-size:12.0pt;font-family:"Aptos",sans-serif;color:black">Borries Demeler <demeler@gmail.com><br>
<b>Date: </b>Sunday, January 7, 2024 at 2:43 PM<br>
<b>To: </b>Gupta, Yogesh K <GuptaY@uthscsa.edu>, Baranwal, Jyoti <baranwal@uthscsa.edu><br>
<b>Cc: </b>demelerlab@biophysics.uleth.ca <demelerlab@biophysics.uleth.ca><br>
<b>Subject: </b>AUC results<o:p></o:p></span></p>
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<p class="MsoNormal" style="background:#FFFFC8"><b><span style="font-size:12.0pt;color:#FF6600">*EXTERNAL EMAIL*</span></b><span style="color:black">
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Hello Yogesh and Jyoti, <o:p></o:p></p>
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<p class="MsoNormal">Kate and Sophia performed your AUC experiment and analyzed the results with all of our tools in our toolbox.<o:p></o:p></p>
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<p class="MsoNormal">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.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"> <o:p></o:p></p>
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<p class="MsoNormal"><img border="0" width="685" height="605" style="width:7.1354in;height:6.302in" id="_x0000_i1029" src="cid:ii_lr3x7xaj0"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><img border="0" width="1079" height="1355" style="width:11.2395in;height:14.1145in" id="_x0000_i1028" src="cid:ii_lr3xjtut1"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">As you can see, all samples return values very close to each other, with a moderate anisotropy of ~ 1.4.<o:p></o:p></p>
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<p class="MsoNormal">The statistics of this fit for the boxed solutes are shown below:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><img border="0" width="536" height="440" style="width:5.5833in;height:4.5833in" id="_x0000_i1027" src="cid:ii_lr3y4zv03"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p style="margin:0in"><b><span style="font-family:"Courier New"">Solute 1:</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Molecular weight: 1.9880e+04 (1.6212e+04, 2.3548e+04)</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Sedimentation coefficient: 1.8990e-13 (1.6992e-13, 2.0988e-13)</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Diffusion coefficient: 8.5097e-07 (7.2433e-07, 9.7760e-07)</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Frictional ratio: 1.4111e+00 (1.2704e+00, 1.5518e+00)</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Partial specific volume: 7.2730e-01 (**constant**)</span></b><o:p></o:p></p>
<p style="margin:0in"><b><span style="font-family:"Courier New"">Partial concentration: 5.9745e-01</span></b><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><img border="0" width="536" height="440" style="width:5.5833in;height:4.5833in" id="_x0000_i1026" src="cid:ii_lr3y99v94"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Furthermore, and overlay of the van Holde - Weischet G(s) plots also indicate perfect homogeneity, reflective of a monomeric species:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><img border="0" width="859" height="1054" style="width:8.9479in;height:10.9791in" id="_x0000_i1025" src="cid:ii_lr3yl2i35"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">All of these plots are uploaded to your LIMS account. Kate will write a report on this experiment and share it with you.<o:p></o:p></p>
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<p class="MsoNormal">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.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Best wishes, -Borries<o:p></o:p></p>
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<p class="MsoNormal" style="background:#FFFFC8"><b><span style="font-size:12.0pt;color:black">CAUTION:
</span></b><span style="font-size:12.0pt;color:black">This message originated outside of UT Health San Antonio. Please exercise caution when clicking on links or opening attachments.</span><span style="color:black">
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