[Demelerlab] [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

[Conticello, Vincent]

Hi, All. I have been busy writing an exam today for a grad course that I am teaching. I am following up on Andres' remarks. It is strange that the 1 mM Calcium ion did not induce assembly of Hyper2, at least compared to Andres' previous experiments. Could it be that some very large species were formed that immediately sedimented? I guess that these species, if they existed, would be visible in the early stages of the experiment. Adding 10 mM Calcium to Hyper2 usually causes immediate precipitation at higher concentration. You could try it-maybe it would not occur at the lower protein concentrations at which you are working. I would think that if incubation for 24 hr with 1 mM Ca caused oligomerization that you would seen its effect over the course of the AUC runs. For CanA, I think that heating to 50C for an hour in the presence of Ca should be fine. Once oligomerization occurs, it is unlikely to be reversible. Regards, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>
Sent: Friday, October 6, 2023 10:55 AM
To: Borries Demeler <demeler at gmail.com>; Martin, Reece <reece.martin at uleth.ca>
Cc: Conticello, Vincent <vcontic at emory.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hello all,

We expected CanA to not polymerize at room temperature with calcium ions. Previous findings suggest that it needs at least 50C incubation for around 1 hour. However, for Hyp2 it was surprising to not see any polymerization. Through TEM I observe tubes when incubated with 1mM Ca at room temperature. I think we could try adding 10mM Ca instead (which causes rapid polymerization) or incubating it for 24hr as previously mentioned. I was wondering if it is a good idea to include a positive control. Terbium (III) causes polymerization almost immediately so it should show a great change in the sedimentation coefficient. What are your thoughts?

Thanks for the update!
Best,
Andres


________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Thursday, October 5, 2023 8:07:49 PM
To: Martin, Reece <reece.martin at uleth.ca>
Cc: Conticello, Vincent <vcontic at emory.edu>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hey guys,
Reece is spot on - there is no change.
Nice work, Reece! Vince, what's your next step?
-Borries


On Thu, Oct 5, 2023 at 5:41 PM Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>> wrote:
Hi Vince and Andres,

      I have analyzed the results from the one-hour incubation with calcium ions (20 mM for CanA and 1mM for Hyper2) at ambient temperature. In order, the four images attached are as follows: CanA High concentration (35.4 μM), CanA Low Concentration (8.3 μM), Hyper2 High Concentration (33.9 μM), and Hyper2 Low Concentration (10 μM). Within each plot, the red trace shows the results from the run with no calcium ions present, while the green trace shows the results from the run with the one-hour incubation with calcium ions at ambient temperature. Therefore, each plot compares a specific concentration of each protein before and after calcium ion incubation. As no significant shift in sedimentation coefficient values were observed, this would imply that none of the species underwent polymerization, at least to an extent that the instrument could detect. Despite this, both proteins thankfully still appear to be intact. In an earlier email you suggested that we could try incubating Hyper2 at ambient temperature for twenty-four hours, and then spin at ambient temperature again. For CanA, you suggested a one-hour incubation at thirty-seven degrees Celsius followed by spinning at thirty-seven degrees Celsius. Let me know how these next steps sound, and we can go from there.

 Cheers,

Reece
________________________________
From: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Sent: 02 October 2023 2:18 PM
To: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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OK, Reece. Thanks for the update. Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>
Sent: Thursday, September 28, 2023 12:54 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi all,

I have completed the experiment with the ambient temperature calcium ion incubation. I am currently working on processing and analysing the data. I will report back next week with the results, and we can decide how we would like to proceed from there.

Cheers,

Reece
________________________________
From: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Sent: 23 September 2023 10:49 AM
To: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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Hi Reece. This sounds great. Regards, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>
Sent: Friday, September 22, 2023 6:07 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi all,

      As Bo mentioned, the first AUC run of both proteins with no calcium ions appears to show that both proteins are still intact and have not aggregated (or polymerized). I am happy to report that the models look good, the RMSDs are fairly low, and the results seem to indicate homogeneity of the protein samples. For the next run I will load the proteins [CanA High conc. (35.4 μM), CanA Low conc. (8.3 μM), Hyper2 High conc. (33.9 μM), and Hyper2 Low conc. (10 μM)] under ambient conditions with the calcium ions (20 mM for CanA and 1mM for Hyper2), program the instrument to incubate the samples for one hour at 20 degrees Celsius before spinning, and then perform the AUC experiment at 20 degrees Celsius. Let me know how this sounds. I will report back once I have the results next week.

Cheers,

Reece
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: 22 September 2023 5:56 AM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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Yes, Vince, there is good news! Reece analyzed the samples we received from you and despite all the complicating factors, they did not show any aggregation. What experiment would you like us to run next? Adding Ca++ or temperature incubation?
-Borries

On Fri, Sep 22, 2023, 05:42 Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi Borries and Reece. I am just following up to see if any further information is needed and if it is OK to proceed with the initial AUC runs. Thanks, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Sent: Saturday, September 16, 2023 1:59 PM
To: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

OK. Sounds like a good control experiment. Best, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Saturday, September 16, 2023 12:37 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Vince et al:
We could run the buffer by themselves to see if the contaminants are just small molecules or something bigger like a protein or DNA fragment.
This way we would have some idea about where this background comes from. Maybe there is something growing in the buffer, or even leaching out from the tubes in which they were stored? Small molecules would not sediment, just give an absorbing background.

-Borries

On Fri, Sep 15, 2023 at 1:11 PM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi All. Andres and I spoke about this issue. We checked the UV-Vis of the buffers that he had made. The EDTA-free buffer does seem to have some contamination, while the EDTA-containing buffer seems much lower. I am not sure if Reece plans to check the spectra of the protein solutions or has done so yet. Andres and I thought that it would be best to proceed forward with the AUC analysis of the pure protein samples (CanA and Hyper2) and then make a decision based on the results of whther the calcium studies should be attempted. Is this agreeable? Best, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Thursday, September 14, 2023 7:33 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi Vince,
protein (280 nm) and DNA (260 nm) absorbance are just one of many possibilities for the source of the spectral properties of your buffer. When we use spectrally pure TRIS or phosphate to make buffers they have a completely flat absorbance at wavelengths > 215 nm. So I am not sure where your buffers picked up the contaminating spectral contributions. If you recall, the samples arrived at RT and the gel packs had leaked over the samples and buffer tubes. I am sure Reece was careful not to contaminate the buffers with the leaked gel material, but all in all the shipping condition was less than ideal. You better check your buffer stocks, maybe that will help identify the source of the contamination.  The absorbance spectrum of the -EDTA buffer is definitely of concern.

We need to know if we should proceed with the experiments under these conditions. As Reece correctly notes, there is a good chance that whatever is contaminating the buffer will sediment with a different s-value than the proteins of interest, but there is a question if the proteins are useful once exposed to contaminants.

Thanks, and sorry for the bad news.

-Borries



On Thu, Sep 14, 2023 at 5:01 PM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Thanks Reece. I will discus with Andres but it is difficult to fathom how the buffers become contaminated with DNA and protein. Regards, Vince

Sent from my iPad

On Sep 14, 2023, at 6:16 PM, Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>> wrote:


Hi Vince and Andres,

      Please see the attached UV/Vis spectrophotometer scans, which were performed against a blank of water. First is the low salt buffer with EDTA, which appears to have nucleic acid contamination. Second is the low salt buffer with no EDTA, which appears to have both nucleic acid and protein contamination. Of course, the proteins are in the EDTA-containing buffer, so this is the one we would be proceeding with. I, of course, have not been able to collect scans of the stock protein samples in a reasonable OD range for the spectrophotometer, so I cannot yet say whether they experience the same nucleic acid contamination.

      My question is whether you would like us to proceed with preparing the samples in the contaminated EDTA-containing buffer. I should note that the nucleic acid contamination should not pose too much of an issue for this experiment. Especially as the nucleic acid contaminant only contributes roughly 0.05 OD at 280 nm according to the spectrum, and would likely sediment different than the proteins, depending on the hydrodynamic properties of the contaminant.

Cheers,

Reece
________________________________
From: Demelerlab <demelerlab-bounces at biophysics.uleth.ca<mailto:demelerlab-bounces at biophysics.uleth.ca>> on behalf of Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: 09 September 2023 2:14 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Subject: Re: [Demelerlab] [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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Hi Vince,
since we collect data typically over 12-15 hours, especially for smaller proteins or peptides, it will be possible to selectively look at early, middle and late data, to identify trends and get some idea about the time dependence. Since we don't know what the answer will be, I am confident we can find out by simply doing the experiment. I just wanted everyone to be aware of the potential limitations of AUC for such a sample.


Thanks, -Borries

On Sat, Sep 9, 2023 at 2:00 PM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi Borries. Yes, I understand what you and Reece are saying. I doubt that these polymerization processes operate under thermodynamic control, i.e., relax to equilibrium at a given temperature after a suitable time period. In related bacterial systems, it is clear that polymerization is kinetically irreversible. However, the kinetics of assembly are slow . Those systems are a bit different in that the normal polymerization process is catalyzed by another protein rather than by metal ion addition. The idea would be to trap, at least on the time frame of the AUC analysis, some early intermediates in the process to gain insight into their hydrodynamic properties.

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Friday, September 8, 2023 12:31 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi Vince,
for AUC experiments to work out really well, it is best if the sample is at chemical equilibrium. If the material changes during the run (e.g., aggregate or degrade), the s value continually changes and can't be fitted well, but for a small slice in time. So the question is not really where we equilibrate for temperature (the AUC instrument can be programmed to pre-incubate the same before the rotor is accelerated, as Reece pointed out), but the main question is for how long such that the incubation leads to chemical equilibrium. I don't think any of us actually knows the answer until we do the experiment. Therefore, I propose that we load the sample under ambient conditions and then program the instrument to pre-incubate at 37 C without spinning for 1 hour and then start the spin. We'll see what happens. If necessary, we can take a fresh sample and increase the incubation time in a second run, or we slice the resulting data into early, middle and late scans and analyze them individually. How does that sound? It would be a good start for this study, regardless of outcome.

-Borries

On Fri, Sep 8, 2023 at 9:44 AM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi Reece. I think that the best approach may be to heat the sample for an hour then run the centrifuge at 37°. Not sure what the best approach would be, i.e., to heat the sample initially outside the centrifuge or inside. I suppose that it depends on what is most convenient for you and/or what might have the least detrimental effect on the instrument. I think that once the sample starts to oligomerize that the process is essentially irreversible. Best, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>
Sent: Thursday, September 7, 2023 5:39 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi Vince and Andres,

Thank you for your responses. I have everything that I need to run the first experiment. I will update once it is completed, and we can go from there.

Vince, in regard to the higher temperature studies with CanA, the maximum temperature the instrument is capable of running at is 40 degrees Celsius. So, we could run the instrument at 37 degrees Celsius with the sample. However, if we were expecting to see oligomerization as a result of heating to (or being held at) this temperature, then we would need to first incubate with calcium ions at 37 degrees Celsius. This could be achieved transiently through a heating block for an hour before sample loading, or more slowly via the AUC as it heats to 37 degrees Celsius over the course of roughly one hour (Bo, please correct me if this is wrong). The AUC then has a temperature-equilibration delay time which we can set. The timescale of the oligomerization would also need to be considered for the temperature-equilibration delay as significant oligomerization during the course of the AUC experiment would yield uninterpretable results. However, if it is the act of cooling to ambient temperature that induces oligomerization, then we would incubate at 37 degrees Celsius with calcium ions for an hour, allow the sample to cool to ambient temperature, and then run at ambient temperature. Perhaps you or Bo have better insight into which of these methods would be more suitable. If not, I can certainly try both.


Cheers,

Reece
________________________________
From: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>
Sent: 07 September 2023 12:58 PM
To: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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Hi everyone,

I just wanted to add the last details. I included some tubes containing the exact buffer in which the proteins are found and another tube with the buffer with no EDTA. In short, the proteins are in a low salt buffer solution (80mM NaCl, 50mM Tris/HCl pH= 7.5, 9% glycerol and 0.01 mM EDTA) thus they all contain 0.01mM EDTA.

Let me know if anything else is needed.
Best,
Andres
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Thursday, September 7, 2023 2:44 PM
To: Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

This sounds good to me. Glad to hear the proteins were not damaged by the elevated temperature storage conditions. We will commence our studies using the samples you provided. The last thing we need to know is what the EDTA concentrations in the buffer are of the samples you provided to us.

-Borries

On Thu, Sep 7, 2023 at 10:55 AM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi Reece. The samples should be good since the proteins are thermostable, that is, from a hyperthermophilic organism even though expressed in E. coli. Andres can answer the first part of your question regarding the buffers since he prepped the samples. For the initial experiments in the presence of calcium, the samples should be incubated at ambient temperature in the presence of calcium ion, probably for an hour. CanA might not oligomerize under these conditions, while the Hyper2 sample should. If the initial calcium experiments do not demonstrate oligomerization, the Hyper2 sample can be incubated for 24 hours at ambient temperature. I would not heat the Hyper2 sample since it might rapidly polymerize (based on our experience). With regard to the CanA sample, I would suggest using the 10 mM or maybe 20 mM calcium ion concentration at higher temperature. If we wanted to detect the presence of oligomers, we might not want to heat the sample to higher temperatures (typically we use 80C for filament formation followed by cooling to ambient to induce polymerization).I think that 37 degrees is a good compromise. Would you run the instrument with the sample at 37 or incubate at 37 and run the sample at ambient? If the latter, it may be best to start out at 1 hr incubation at elevated temp. Best, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>
Sent: Wednesday, September 6, 2023 7:11 PM
To: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi Vince and Andres,

Considering that the samples arrived at room temperature (the exact length of time is unknown), and that an ice pack leaked (the samples appear to have been sealed properly). We are just looking to confirm that you feel it is worth continuing to process this batch of samples, taking into account your insight into the temperature-stability of the proteins at ambient temperature.

If so, as per the updated experimental design in the LIMS, we will start out running both CanA and Hyper2 at a high and low concentration in the EDTA-containing buffer at 20 degrees Celsius. To accomplish this, I need to know what buffer each of the protein stocks are in and whether or not they contain EDTA.

If this experiment is successful, we will repeat in the presence of calcium ions (10mM for CanA, and 1mM for Hyper2). For this we need to know at what temperature and for how long the samples should be incubated with the CaCl2 solution. Finally, if this experiment is successful, we will look at repeating with increased calcium ion concentrations, as well as running at a higher temperature such as 37 degrees Celsius.

I'll report back on the results from the first run, which will take place this week.

Cheers,

Reece

--
Reece Martin, B.Sc
M.Sc. Candidate
Northwest Biophysics Consortium
Alberta RNA Research and Training Institute (ARRTI)
Lab of Dr. Borries Demeler
Department of Chemistry and Biochemistry
University of Lethbridge
Email: reece.martin at uleth.ca<http://uleth.ca/>
Phone: 403-498-5882
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