Happy New Year! Excited to share the final version of our work where we combine deep mutational scanning, cryo-EM, and molecular dynamics to further resolve the mechanisms of pH sensing in GPCRs. 🧵(1/n).

RT @willowcoyote: Check out our work, where we discover how receptor GPCR senses pH! We develop foundational GPCR DMS tech dev + structural….

Thanks for the invite! Excited to share some of our recent work tomorrow 😀.

Tagging all the folks who I can find here. @willowcoyote @AashishManglik @JGEnglishLab @DelemotteLab @Darko_physics @ahuangxp @LleBillesb @protostasis.

RT @zenbrainest: Amazing study--one of two on proton sensing receptors @CellCellPress . Molecular basis of proton sensing by G protein-coup….

There is certainly more than can be shared here, but I will end it there with a HUGE thanks to everyone involved! This was both a challenging and fun project to be involved in with many great folks without which none of this would be possible. (13/13).

Ultimately, this enabled us to identify residues which change pronation with conformation and pH. Combining our DMS, cryo-EM, MD, and extensive pharmacologic validation (by @ahuangxp), we were able to devise a comprehensive model for pH activation of GPR68 (12/n)

Well, Darko went above and beyond. He developed a new method which uses coevolutionary information to identify both alternative receptor conformations AND the effect of pH on the conformational ensemble. (11/n)

We needed an inactive-state structure model to fully interpret our results, so we teamed up with @Darko_physics (Delemotte Lab) to do some molecular dynamics simulations of GPR68 (10/n).

We followed up with a surface expression screen to determine the which mutations alter receptor expression. This allowed to deconvolve the mutational effects on expression vs activation to uncover GOF and LOF activity specific to activation. (9/n)

The result: a comprehensive map of each mutation's effect on pH activation. (8/n)

We then generated a mutational library of GPR68 using the DIMPLE platform developed in @willowcoyote's lab and screened it using our cAMP FACS assay (7/n)

Using GPR68 as our model, we set out to use mutational scanning to determine the effect of every mutation on proton activation. We developed a new FACS-based method to measure Gs coupled receptor activation (enabled by a new TRE's from @JGEnglishLab)(6/n)

We needed to ascribe a functional role for each residue in proton activation. Unfortunately, these receptors are littered with an enormous number polar and charged residues which may be implicated. (5/n)

To start, Nick Hoppe (in @AashishManglik lab) set out to determine the 3D architecture of each human proton sensor (GPR4, GPR65, and GPR68) using cryo-EM. These provided insights into the architecture and arrangement of putative proton-sensing residues in the active-state (4/n)

We wanted to determine the location and identities of all residues involved in coordinating protons to drive receptor activation. (3/n).

We have known for some time that several GPCRs respond to changes in pH. However, unlike small molecules, peptides, and other stimuli, protons are a bit wonky--we can't directly see them with standard approaches. (2/n)

RT @genophoria: Check out our latest research by @MNaghipourfar and others in our team on building language models of coding sequence. In t….

RT @BiologyAIDaily: A Suite of Foundation Models Captures the Contextual Interplay Between Codons @arcinstitute . 1. This paper introduces….

RT @cool7TMs: 📢#GPCR ECIs, mark your calendar for the next Transatlantic GPCR Symposium! .The event will happen on September 5-6th, 2024 an….