[Demelerlab] AUC (sedimentation equilibrium experiments)

Wed Dec 13 07:28:19 MST 2023

Dear Dr. Demeler,
Good morning!
Thank you for your comprehensive details related to experiments. I will perform the requisite, measure the OD, make an account, and will upload as well.
There is an important information I would like to share with you, I have never seen any visible precipitate or turbidity in any buffer conditions for this protein, and the 8 different buffer and salt conditions I have mentioned in my previous email have shown good peak when I performed for SEC-MALS. The buffers where it wasn't very well behaving are in very low salt conditions which I haven't sent you the details of yet. I am planning to try these buffers only if these 8 buffer conditions don't work.
I would like to clarify a doubt I have; would you want me to perform aggregation test for these 8 buffers I have mentioned (where I haven't seen any sign of precipitation)? Because in my experience this protein is pretty stable in these buffers and, we only want to see if there is any oligomeric state. However, if it helps in performing AUC, I will be happy to check aggregation in all the buffers.

My sincere appreciation,

With best regards,
Jyoti Baranwal
________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Tuesday, December 12, 2023 4:17 PM
To: Baranwal, Jyoti <baranwal at uthscsa.edu>
Cc: Gupta, Yogesh K <GuptaY at uthscsa.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: AUC (sedimentation equilibrium experiments)

*EXTERNAL EMAIL*

Hello Jyoti,
Thanks for the details in your message. It sounds like you may have some issues with aggregation in certain buffers. There is a very simple test you can do if you think this is an issue:

1. measure the absorbance spectrum of your protein from ~ 220-600 nm. If you see non-zero absorbance above 300 nm, you likely have aggregation.
2. put your protein solution in a benchtop microfuge and spin for 3-4 minutes. Carefully aspirate the supernatant and re-measure the absorbance as above. If the absorbance is noticeably lower, then you lost material due to aggregation. Also, make sure the non-zero absorbance >300 nm is gone. That portion is your aggregate.

If the buffers below produce no aggregate, then we can help you measure reversible oligomerization. I am not familiar with MBis, but I do believe acetate buffers absorb below 240 nm, so there we need to know how much. The PBS buffer should not absorb at all in this range if it is clean. The TRIS buffers should be fine as well, as long as you use optical grade TRIS. Also, 50 mM is probably more than you need, so you could go down to 10-15 mM TRIS without losing buffering capability for a low concentration protein. For the acetate buffers I suggest to go to low enough concentration to maintain an absorbance of < 0.3 OD at the lower wavelength where you will want to measure the lower protein concentrations.

It would be good if you could collect buffer absorbance profiles and send them to us for review so we can ascertain optimal conditions.

How much protein do you have available to make these measurements? I would suggest we measure 0.3 OD at 230 nm, and 0.7 OD at 285 nm for each buffer so we can get a good spread of concentrations. Can you please calculate the molar concentrations at these wavelengths and send them to us? You can get a molar extinction coefficient for 280 nm if you upload the sequence into UltraScan's analyte management tool - Yogesh should know how to do that, or we can do it for you.

Please create a LIMS account on our server so we can track this project at this address:
https://uslims.uleth.ca/uslims3_CCH/newaccount.php
Once you created the account, please check your e-mail for an activation link and a temporary password. You can update it after logging in under "Change my info". Next, please submit a project request ("Projects" button in the left bar), and answer questions in the form as best as you know. Leave the experimental design section blank, we will fill this in. Don't forget to include the protein sequence and buffer components. You can also upload PDF documents or images with the absorbance spectra to our LIMS server by clicking on "Supporting Files".

Once this information is uploaded, we will work out a design and we can schedule a zoom call to discuss these experiments further.

Regards, -Borries

I have used a list of buffers to check its oligomeric state of the protein using SEC-MALS. However, there could be possible that salt bridges between the protein is not stable in the SEC-MALS, that's why we aren't able to observe the dimer/oligomeric state of the protein and only get the peak for monomer. We have observed some fraction of protein at higher oligomer when we have lowered the salt concentration in the buffer, but protein seem to be unstable.
We think that AUC would be very helpful to understand the oligomeric state of this protein. Therefore, we want to try different pH, and salt concentration.  We can couple this with multiple concentration of protein, as you have suggested.
I have made a list of buffers we would like to try with this protein (listed below). All the buffer solution has only buffering agent and salt in it. There is no other compound which will hinder absorbance.

  1.  200 mM LiSO4 100 mM Sodium acetate pH 4.5
  2.  50mM Tris-HCl 50mM KCl pH7.5
  3.  1x PBS
  4.  25mM Sodium acetate 100 mM NaCl pH 4.5
  5.  25mM MES 100mM NaCl pH 5.5
  6.  25mM MBis-Tris 100mM NaCl pH 6.5
  7.  25mM Tris-HCl 100mM NaCl pH 7.5
  8.  25mM Tris-HCl 100mM NaCl pH 8.5

On SEC-MALS I have tried two different concentrations 8 mg/ml and 13 mg/ml and 40 ul. To begin with we can start with these buffers and check the oligomeric state if required I have few more buffers to try. We can start with low concentrations of protein and then move to higher concentration. At this stage I can't comment on what concentration we can use for AUC. But what I can say at this point, I have used 200 nM protein on Mass spectrometer it did suggest that it is dimer, but this had a limitation of 30 kDa and because my protein is approx. 19.5 kDa we could not see monomers. Based on the sensitivity of AUC we can decide on the concentration.

Thank you for your time and consideration!

With best regards,

Jyoti Baranwal

Postdoctoral Researcher
Gupta Lab
Greehey Children's Cancer Research Institute (GCCRI)
University of Texas Health Science Center at San Antonio
Email: baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Monday, December 11, 2023 3:34 PM
To: Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>>
Cc: Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>>
Subject: Re: AUC (sedimentation equilibrium experiments)

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*EXTERNAL EMAIL*

Hi Yogesh,
thanks for your message! Yes, this sounds very interesting, so we are interested in testing the effect of pH on the kd and the oligomeric state?
I suggest we measure multiple concentrations, perhaps varying 5-20 fold, above and below the putative kd concentration, at each pH you want to test. Any chance we can access wavelengths around 225-230 nm without buffer background? Any buffer component that absorbs should be kept to a minimum concentration so we don't saturate the dynamic range of the detector.

For each sample, we would need about 0.5 ml volume, optical densities at each wavelength should ideally be between 0.3-0.7 OD.

Before we proceed we should have a meeting with our AUC lab members and discuss this research a bit further.

Thanks! -Borries

On Sat, Dec 9, 2023 at 4:51 PM Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>> wrote:

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Hi Borries,

I hope you are doing well. Jyoti (cc’ed) is a postdoc in my lab who solved the crystal structure of a phosphatase/RNA de-capping enzyme (monomeric molecular mass = ~20 kDa). We observed a dimer in the crystals. Using the sedimentation equilibrium method, we want to check its oligomeric state in solution at different pH  (4.5 – 8.5). Would you be interested in this collaboration? We learned that our AUC in RAB is down, so we didn’t attempt these experiments. We thought you might be interested in this project.

Best,

Yogesh

Yogesh Gupta, Ph.D.
Associate Professor
University of Texas Health Science Center at San Antonio

Department of Biochemistry and Structural Biology

Greehey Children's Cancer Research Institute
8403 Floyd Curl Drive, MC7784

San Antonio, TX, 78229, USA
Phone: 210-562-9064<http://voice.google.com/calls?a=nc,%2B12105629064><http://voice.google.com/calls?a=nc,%2B12105629064>
Gupta Lab<http://ccri.uthscsa.edu/YGupta.html>

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