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

[Borries Demeler]

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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