Videos for registered attendees of the SMALP Conference on March 12, 2024
Ci Chu, PhD student, Heinrich-Heine-University, on capturing GPCRs into native lipid-bilayer nanodiscs using DIBMA copolymers.
Michael Erkelenz, Chemist, Cube Biotech on A new multimodal memtein copolymer platform.
Jelger Risselada, Dept of Physics, TU Dortmund University, on Membrane facial recognition: How proteins actually see cholesterol.
Marzieh Tabefam, Ph. D. student, WLU, on exploring theiInterconnection of Mitochondrial Carrier Proteins Involved in Energy Transfer.
Ayyalusamy Ramamoorthy, Professor, Florida State University, on detergent-free membrane protein isolation using nanodisc-forming polymers.
Elissa Moller, PhD student, NICHD, on using cryo-EM and polymers to resolve structures of mechanosensitive channels.
Peter Kim, Ph.D. Student, University of Toronto, on structures of channel rhodopsin paralogs in peptidiscs and their ion selectivities.
Wayland Cheng, Associate Professor, Washington University, on nanodisc scaffold and size altering ligand-gated ion channel structures.
Brian Adair, Instructor at Harvard Medical School, on cryo-EM structures of human integrins in native lipids.
Chat conversations in the SMALP webconference on March 12, 2024:
01:04:08 Michael Overduin: Feel free to ask Ci Chu questions here.
01:05:58 Hector Ariel Roldan, Dr: Is mPeg4-dibma electroneutral?
01:07:00 Jen: what temperature was the solubilization performed at?
01:08:20 Philipp Hanisch (Cube): Is there in example where Ligand binding of a GPCR failed in other polymers but worked in your new polymer?
01:08:34 Ruby Huynh: Why did you choose DIBMA over other polymer?
01:09:34 Tamara Miljus: Thank you for the nice talk! Have you checked if your receptor can be activated in DIBMA or your new polymers?
01:10:26 Nathan K: Can you provide a link to the paper?
01:12:41 Eve: Thank you, from your research do you suggest we use negatively charged polymer or electroneutral polymers for membrane proteins? Also, did you try other lipids in your study asides DMPC?
01:13:44 Michael Overduin: Next SMALP conference will be by zoom on Tuesday June 18, 2024
01:16:32 Gestél Kuyler: Reacted to "Next SMALP conferenc..." with 👌
01:17:29 d.wright: Reacted to "Next SMALP conferenc..." with 👍
01:17:40 Manuel Etzkorn: Here the link to Ci’s preprint, enjoy reading and feel free to comment: https://www.biorxiv.org/content/10.1101/2024.01.20.576420v1
01:18:08 Alexej Kedrov: Reacted to "Here the link to Ci’..." with 👍
01:18:23 stemuench: Reacted to "Here the link to Ci’..." with 👍
01:24:13 Manuel Etzkorn: Replying to "Thank you, from your..."
We see higher solubilisation yield for our protein in the new mPEG version. The other parameters we tested are similar in sulfo-DIBMA. We used DMPC and DMPG for im vitro assembly.
01:24:42 Praneetha Sundar Prakash: Reacted to "Here the link to Ci’..." with 👍
01:24:53 Praneetha Sundar Prakash: Reacted to "Next SMALP conferenc..." with 👍
01:26:23 Eve: Replying to "Thank you, from your..."
ok... was there a difference in DMPC and DMPG lipid bilayers due to charge of the mPEG dibma charge?
01:29:51 Jana Susanne Anton: How come the absorption is so low in the SEC (slide 17). We were observing similar absorptions in the past with SMA.
01:30:14 Rajesh: Reg Biotin-Cubipol: Thank you for adding Biotin moiety. Is the biotin at 1:1 ratio with PIMA, and do you have any examples of moles biotin in an extracted protein wrapped with Biotin-Cubipol.
01:30:48 Katherine Zhao: Reacted to "Here the link to Ci’…" with 👍
01:31:53 Alice Rothnie: What is the ratio of fluorophore to polymer?
01:33:52 Doreen Matthies: Can you run your purified G6PC on Native-PAGE?
01:36:59 Michael Overduin: Feel free to ask Jelger questions here.
01:37:18 Michael Erkelenz: Replying to "What is the ratio of..."
The Ratio of fluoresceine to Copolymer is 1. So one fluorophor per chain. This ensures no cluster forming by the hydrophobic fluoresceine.
01:37:31 Alice Rothnie: Reacted to "The Ratio of fluor..." with 👍
01:38:33 Michael Erkelenz: Replying to "Reg Biotin-Cubipol: ..."
We will presumably Show more data related to this Topic at our next masterclass. Of Course, you are invited to have a look
01:39:16 Philipp Hanisch (Cube): Replying to "Can you run your pur..."
I jump in for Michael Erkelenz here: We did not try to run G6PC in specifically on Native Page but did this with several other targets in a broad range of polymers and detergents. In my personal expirience it can be quite tricky to get reliable data even when using the published SMA PAGE Approach so i am not a huge fan of Native Page when working with Membrane Proteins. What are your expiriences?
01:39:27 Michael Erkelenz: Reacted to "I jump in for Michael..." with 👍
01:40:37 Philipp Hanisch (Cube): Replying to "Reg Biotin-Cubipol: ..."
the masterclass will be held on 10. or 11.4.24. you can soon Register via our Homepage :D
01:41:43 Doreen Matthies: Replying to "Can you run your pur..."
We have issues with getting some of our polymer samples into NATIVE-PAGE but have no problem when using detergents or MSPs. Elissa will mention that later also and would like to understand why.
01:49:04 Ayyalusamy Ramamoorthy: Is the cholesterol binding independent of the lipid composition?
01:49:30 Ayyalusamy Ramamoorthy: Do curvature inducing lipids influence cholesterol binding?
01:49:51 Renata Bilewicz: what about cholestenone (product of oxidation) interactions in the membrane?
01:50:08 MARTA BARNIOL XICOTA: Did you identify any proteins containing this CHO-attracting sequence and if so, are there any CHO-interactions reported with that protein?
01:50:44 Rajesh: How does your optimal TM domain compare to existing protein database on single pass TM proteins
01:54:09 Jelger: Herre Jelger risselada
01:57:10 Michael Overduin: Feel free to ask Marzieh questions here.
02:00:58 C. Chu: Replying to "Thank you, from your..."
Thanks, we also tried POPG and POPC. All worked well. We only used DMPC for differential scanning calorimetry (DSC) measurement.
02:01:55 Rajesh: Trivia for SMALP attendees: Wilfrid Laurier was Michael Overduin's Alma Mater for Undergrad!😀
02:02:13 Debbie Hansen: Reacted to "Trivia for SMALP att..." with 👍
02:04:11 Michael Overduin: WLU in Waterloo, Ontario, Canada is hosting a free protein symposium on May 31, with John Rubinstein giving the keynote. See https://www.eventbrite.ca/e/tri-university-protein-symposium-tups-2024-tickets-826825977757
02:04:16 Doreen Matthies: Was the tetrameric form SDS stable or did you have to crosslink?
02:04:36 Michael Overduin: Feel free to advertise jobs and student opportunities here.
02:05:11 Doreen Matthies: How did the detergent solubilized proteins look on NATIVE PAGE or SEC?
02:10:14 Doreen Matthies: Replying to "Was the tetrameric f..."
I see now, it is semi-NATIVE PAGE
02:11:20 Doreen Matthies: Can you pull down heterotetramers from the inner mitochondrial membrane?
02:13:45 Andres Cabezas: Did you use polymers at all during the initial protein purification steps? I know ultimately you used detergent to solubilize the membrane proteins, I am wondering if you can use detergent with polymer-solubilized proteins right before the proteoliposome reconstitution step.
02:15:03 Tomáš Heger: Replying to "Was the tetrameric f..."
Could you please explain what you mean by "semi-native"?
02:17:25 Michael Overduin: Questions for Rams?
02:18:16 Marzieh Tabefam: Replying to "Was the tetrameric f..."
Yes, semi-native condition has less SDS percentage on the gel compare to the denaturing condition, and no SDS in loading buffer and running buffer. Does this what you ask for?
02:19:00 Tomáš Heger: Reacted to "Yes, semi-native con..." with ❤️
02:19:12 Tomáš Heger: Replying to "Was the tetrameric f..."
Yes, thank you!
02:28:23 Alexander Nevzorov: Rams: Are the peptides indeed pointing perpendicular to the membrane surface according to your picture? That would appear to break the “belt” hypothesis.
02:29:08 Marzieh Tabefam: Replying to "Did you use polymers..."
Yes! detergent is kept all the way along the reconstitution process and it is removed by bio-beads at the step right before the proteoliposomes formation
02:32:17 Andres Cabezas: Replying to "Did you use polymers..."
If I understood correctly, did you use polymers to natively extra proteins from the e.coli membranes, and then introduced detergent to further solubilize the proteins and perform the subsequent proteoliposome steps?
02:40:23 Michael Overduin: Feel free to ask Elissa questions here.
02:40:28 Marzieh Tabefam: Replying to "Did you use polymers..."
No! we didn't use polymers in our work
02:40:40 (Rams) Ayyalusamy Ramamoorthy: Replying to "Rams: Are the peptid..."
Peptides are amphipathic and helical when bound to lipids as in nanodiscs. They are located at the rim of the nanodiscs with the hydrophobic face facing lipids chains.
02:40:49 (Rams) Ayyalusamy Ramamoorthy: Replying to "Rams: Are the peptid..."
Good question, Alex. Thanks.
02:41:20 Evelyn Okorafor: Rams: Did you test your charged polymers with charged lids asides dmpc?
02:41:44 Andres Cabezas: Replying to "Did you use polymers..."
Got it. Thank you! I wanted to make sure I didn’t miss something. I am doing work similar to yours. Nice talk 🙂
02:41:56 Evelyn Okorafor: Replying to "Rams: Did you test y..."
Rams: Did you test y...
02:42:19 Marzieh Tabefam: Replying to "Did you use polymers..."
Nice. thank you🙂
02:43:11 (Rams) Ayyalusamy Ramamoorthy: Replying to "Rams: Did you test y..."
We did with DMPG, POPC, POPG, E.coli lipids, etc. But, most of these data were not published.
02:46:05 Evelyn Okorafor: Replying to "Rams: Did you test y..."
Alright... Thank you...
02:46:31 Rajesh: Melissa: Do yo expect similar differences with lipid composition influencing structure and function of eukaryotic Aquaporins?
02:52:20 LG Zeng: Melissa: what were the chain length of your lipids?
02:52:23 Michael Overduin: Melissa: Did the multiple lipids bind as a set that may have been disrupted by the other polymers? Glyco-DIBMA is gentle.
02:52:39 Rajesh: Reacted to "Melissa: Did the mul..." with 👍
02:59:59 Michael Overduin: Feel free to ask Peter Kim questions here.
03:09:48 LG Zeng: Peter: I might have missed it. What was the number of amino acids/molecular weight of the peptides for your peptidiscs? And what is the size of the resultant discs? Thx.
03:12:08 Aeric Yue Zhou: Do all three protomers have to be activated before letting the K+ or Na+ go through?
03:13:18 Rajesh: Peter: That's a lot of fat (cholesterol) bound to KCR1🙂. Is it understood why Chol is tightly bound despite detergent extraction?
03:14:43 Michael Overduin: Peter: can you comment more on the nature of the protein-lipid head group and acyl chain interactions and the protein-peptidisc interactions?
03:22:08 Michael Overduin: Feel free to to ask Wayland questions here.
03:22:35 Peter (Kyumhyuk) Kim: Replying to "Peter: I might have ..."
The peptide length is 37 amino acids, and the molecular weight should be close to 4.5kDa
03:33:55 Rajesh: Wayland: Really enjoyed reading your paper. Were you able to see any bound lipids. Is there any preference for particular lipids with the different reconstitution formats.
03:38:52 Michael Overduin: Wayland: what are the scaffold interactions, e.g. scaffold aromatics pi interactions with transmembrane residues?
03:39:35 Johanna Syrjanen: Wayland: Is the positioning of ELIC to the side of the nanodisc affected by the lipid composition of the nanodisc?
03:39:47 Michael Overduin: Are there MSPs where the aromatic residues have been successfully eplaced with long chain aliphatic residues?
03:41:22 Tomáš Heger: Is the preferred localization of ELIC caused solely by interaction with MSP or does the membrane thickness also contribute?
03:41:24 Alexander Nevzorov: Great talk! Could you clarify why you observe membrane thickening for smaller disks?
03:42:18 Alexander Nevzorov: Is it protein-induced?
03:43:13 Ilia G Denisov: There is significant difference between nanodiscs and LUV. But real membranes contain 20%- 60% proteins by weight, is this a factor to consider?
03:49:44 Michael Overduin: Feel free to ask Brian questions here.
03:50:18 Michael Overduin: The student prize will be awarded after this talk. Thanks for voting!
04:01:02 Doreen Matthies: Did you try to collect Cryo-EM data at a tilt angle and did this improve your reconstruction?