[Demelerlab] Fwd: AUC Collaboration-Pecoraro Lab

[Borries Demeler]

Reece/Sophia/Aysha:

We are working with Vince Pecoraro and Winston to study Cu-binding
peptides, please see below for a discussion on Sept. 19th at 1 pm:


---------- Forwarded message ---------
From: Winston Pitts <wcpitts at umich.edu>
Date: Tue, Aug 29, 2023 at 7:58 PM
Subject: Re: AUC Collaboration-Pecoraro Lab
To: Borries Demeler <demeler at gmail.com>
Cc: Vincent Pecoraro <vlpec at umich.edu>


Hey Borries,

Thank you for the info! Let's do either Tuesday (9/19) or Thursday (9/21)
at 3pm est (1pm MT). Which one works best for you?

Best,

Winston C. Pitts
PhD Candidate, Inorganic
Pecoraro Group
Department of Chemistry
The University of Michigan



On Sun, Aug 20, 2023 at 12:05 PM Borries Demeler <demeler at gmail.com> wrote:

>
> My name is Winston, and I'm a 5th year PhD student with Vince. We are
>> looking to answer some questions about the oligomeric states of some of my
>> designed proteins (Figure 1).
>>
>> Hi Winston, nice to e-meet you!
> FYI, it looks like your Figure 1 did not make it into the e-mail, and
> there was nothing attached either.
>
>
>> I'm working with homotrimers that have non-coded heterocycles in
>> their active sites (pyridine in this case) that bind copper in order to
>> explore their copper nitrite reductase activity. It turns out that these
>> active sites are more efficient at catalytic NO2- to NO turnover than
>> histidine, and when coupled to an L19A mutation (directly above the
>> active site), the 4'pyridine active site becomes the fast CuNiR mimic
>> currently reported (kcat/Km 39 M-1 s-1) in water including those that are
>> small molecule mimics on an electrode surface.  From the steady state
>> kinetics, this is primarily driven by a decrease in Km when alanine
>> replaces leucine in the 19 position.  We have evidence that NO2- binds
>> directly to the Cu(I) oxidation state making the geometry around Cu(I)
>> important for substrate binding.  When this alanine mutation is
>> present with histidine, we shift to a bis-His Cu(I) (XANES/EXAFS) from the
>> 3-coordinate Cu(I) site when leucine above. However, the pyridine's Cu(I)
>> XANES/EXAFS with the alanine suggest that we have a mixture of
>> 3-coordinate and 2-coordinate Cu(I) for both 3'pyridine and 4'pyridine
>> (both models fit the EXAFS within error of the data). While there are
>> at least three possible explanations I have come up with to explain what it
>> means to have "mixed" coordination modes in the XANES/EXAFS , one possible
>> explanation is that there is a mixture of 2SCC and 3SCC.
>>
>
> I believe you are referring to dimer/trimer configurations?
>
>
>> **What I want to know is whether or not I have 3SSC/2SCC/monomer over the
>> concentration range where I conducted my various experiments (1uM
>> trimer-Kinetics, 300uM trimer UV-VIS, 1.5 mM trimer for both Cu XANES/EXAFS
>> and EPR). I have UV-VIS experiments showing that Cu(II) binds with a 1:1
>> Cu(II)/Trimer ratio at 300uM trimer, and Cu(I) NMR at 3 mM trimer showing a
>> 1:1 Cu(I)/Trimer for the peptides I have been able to get done. However, I
>> have really no information on the lower concentration region where I
>> conduct my kintices (1uM trimer). I believe AUC would be helpful somewhere
>> in the vicinity of these concentrations. I'm not sure what signal you would
>> want to look at for the lower concentrations, but I have a Trp. near the
>> N-terminus (e280=5,500 M-1 cm-1 monomer-1) that will give you an Abs of ~
>> 0.016 at 3uM monomer (1uM trimer).
>>
>
> We should be able to get close to the molar mass of the predominant forms.
> Given the relatively large concentration range you are exploring, I think
> it would be wise to measure at multiple concentrations to obtain a mass
> action profile for this protein in case it is self-associating. Given the
> small molar mass, we would have to measure at very high speeds (60 krpm) to
> resolve monomer/dimer/trimer forms.
>
> 0.016 OD is too little to get enough signal, we would need at least 0.3
> OD, 0.6 OD would be optimal. If you can measure in a non-absorbing buffer -
> phosphate or optically pure TRIS would be ideal with low salt (ca. 10-50
> mM) would be ideal - we could also measure the backbone peptide bond
> absorbance (~ 300 OD/mol) between 220-225 nm, which could be a bit higher
> than the W absorbance. We could measure a decent concentration range where
> the OD is 0.3-0.9 OD at different wavelengths to explore the mass action
> potential.
>
> Of course we cannot help with the functional aspects of your study, also,
> the kinetics are way too fast for our system to measure, so we cannot help
> with this, but the oligomerization question is quite straightforward to
> answer.
>
> If you would like to have a virtual meeting next week to go over things
>> in more detail, let me know what times work best for you, and we can work
>> it out.
>>
>
> I would like to have a meeting with you guys and my team to discuss the
> project we would be doing. The next three weeks are very bad for meetings,
> as we run our annual AUC workshop, student thesis defenses, and the
> semester start, which is always very busy. After the 17th would be good.
> Feel free to schedule a zoom call for that week. I will be available most
> afternoons (Mountain Daylight Time).
>
> Best wishes, -Borries
>
>>
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