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

[Borries Demeler]

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> 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>
> *Sent:* 02 October 2023 2:18 PM
> *To:* Martin, Reece <reece.martin at uleth.ca>; Gonzalez Socorro, Andres <
> andres.gonzalez.socorro at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <demelerlab at biophysics.uleth.ca>
> *Subject:* Re: [External] Analytical Ultracentrifugation Analysis of
> Cannula-mimetic Proteins
>
>
> Caution: This email was sent from someone *outside of the University of
> Lethbridge*. Do not click on links or open attachments unless you know
> they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>
> 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>
> *Sent:* Thursday, September 28, 2023 12:54 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>; Gonzalez Socorro, Andres <
> andres.gonzalez.socorro at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <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>
> *Sent:* 23 September 2023 10:49 AM
> *To:* Martin, Reece <reece.martin at uleth.ca>; Gonzalez Socorro, Andres <
> andres.gonzalez.socorro at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <demelerlab at biophysics.uleth.ca>
> *Subject:* Re: [External] Analytical Ultracentrifugation Analysis of
> Cannula-mimetic Proteins
>
>
> Caution: This email was sent from someone *outside of the University of
> Lethbridge*. Do not click on links or open attachments unless you know
> they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>
> 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>
> *Sent:* Friday, September 22, 2023 6:07 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>; Gonzalez Socorro, Andres <
> andres.gonzalez.socorro at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <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>
> *Sent:* 22 September 2023 5:56 AM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>; 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
>
>
> Caution: This email was sent from someone *outside of the University of
> Lethbridge*. Do not click on links or open attachments unless you know
> they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>
> 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> 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>
> *Sent:* Saturday, September 16, 2023 1:59 PM
> *To:* Borries Demeler <demeler at gmail.com>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>; 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
>
> 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>
> *Sent:* Saturday, September 16, 2023 12:37 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>; 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
>
> 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>
> 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>
> *Sent:* Thursday, September 14, 2023 7:33 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>; 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
>
> 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>
> 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> 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> on behalf of
> Borries Demeler <demeler at gmail.com>
> *Sent:* 09 September 2023 2:14 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>;
> Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>
> *Subject:* Re: [Demelerlab] [External] Analytical Ultracentrifugation
> Analysis of Cannula-mimetic Proteins
>
>
> Caution: This email was sent from someone *outside of the University of
> Lethbridge*. Do not click on links or open attachments unless you know
> they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>
> 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>
> 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>
> *Sent:* Friday, September 8, 2023 12:31 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* 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
>
> 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>
> 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>
> *Sent:* Thursday, September 7, 2023 5:39 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>; Gonzalez Socorro, Andres <
> andres.gonzalez.socorro at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <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>
> *Sent:* 07 September 2023 12:58 PM
> *To:* Borries Demeler <demeler at gmail.com>; Conticello, Vincent <
> vcontic at emory.edu>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>;
> demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
> *Subject:* Re: [External] Analytical Ultracentrifugation Analysis of
> Cannula-mimetic Proteins
>
>
> Caution: This email was sent from someone *outside of the University of
> Lethbridge*. Do not click on links or open attachments unless you know
> they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>
> 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>
> *Sent:* Thursday, September 7, 2023 2:44 PM
> *To:* Conticello, Vincent <vcontic at emory.edu>
> *Cc:* Martin, Reece <reece.martin at uleth.ca>; 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
>
> 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>
> 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>
> *Sent:* Wednesday, September 6, 2023 7:11 PM
> *To:* Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>;
> Conticello, Vincent <vcontic at emory.edu>
> *Cc:* Borries Demeler <demeler at gmail.com>; demelerlab at biophysics.uleth.ca
> <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
> Phone: 403-498-5882
>
> <LowSaltBufferwithEDTA_contaminant.jpg>
> <LowSaltBufferwithNOEDTA_contaminant.jpg>
>
>
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