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Real-Time Tracking of Membrane Protein Insertion Using Dynamic Mass Photometry

Secondary Supervisor(s): Dr Manish Kushwah, Dr John Simms

University of Registration: Aston University

BBSRC Research Themes:

• Understanding the Rules of Life (Structural Biology)

Project Outline

Integral membrane proteins (IMPs) are synthesized on endoplasmic reticulum (ER)-bound ribosomes and cotranslationally inserted into the ER membrane from the N- to C-terminus, aided by membrane integration machinery. However, the details of this process remain poorly understood, largely due to the lack of methods for real-time monitoring of membrane protein synthesis and insertion at the level of individual ribosomes. This gap hinders our understanding of folding pathways, which is crucial for addressing protein misfolding and developing therapeutics targeting rare, transient cotranslational intermediates.

To visualise the highly dynamic process of cotranslational IMP biogenesis, we will follow nascent chain elongation in real time at a single molecule level using in vitro expression systems with artificial ER bilayer mimics, and dynamic mass photometry (DMP); a state-of-the-art, label-free, mass photometry-based, single-particle-tracking method that accurately measures molecular weights and diffusion of proteins diffusing on supported bilayers (SLBs). This modular system also allows the lipid composition of bilayers to be tuned, enabling the investigation of how specific lipid chemistries influence membrane protein insertion and folding.

1) Investigate spontaneous and translocon assisted insertion of model human IMPs into synthetic lipid vesicles.

2) Use artificially stalled ribosome-nascent chain complexes (RNCs) of defined lengths to benchmark membrane insertion using DMP.

3) Monitor cotranslational IMP insertion in SLBs by observing mass and diffusion change using DMP.

4) Vary lipid bilayer composition to probe the influence of lipid environment on membrane protein insertion efficiency.

5) Incorporate recombinant chaperones and integration machinery into bilayers to assess their effect on insertion dynamics via DMP.

Pellowe & Booth, 10.1016/j.bbamem.2019.07.007

Pellowe et al, 10.1021/acs.biochem.0c00423

Foley, Kushwah et al, 10.1038/s41592-021-01261-w

How do aquaporin water channels fold and insert into the membrane?

Secondary Supervisor(s): Dr Philip Kitchen, Prof. Roslyn Bill

University of Registration: Aston University

BBSRC Research Themes:

• Understanding the Rules of Life (Structural Biology)

Project Outline

Membrane proteins are essential for cellular function and as drug targets. The aquaporin (AQP) family of membrane proteins, which are crucial for water transport across all cell membranes, rely on accurate folding and localisation for their function. Misfolding of AQPs is linked to severe conditions of the brain (AQP4), kidney (AQP2) and lung (AQP1).

Very little mechanistic information exists about the folding and insertion of membrane proteins during their synthesis on the ribosome. Intermediate folding states are poorly characterised, and no method currently enables the capture of folding intermediates from the human endoplasmic reticulum (ER) in sufficient quantities for advanced biophysical analysis.

This exciting project will use original methodologies to investigate the biogenesis of AQPs at the ER during synthesis, aiming to pinpoint when and why misfolding occurs and how that can be used to understand human disease.

1. Capture and purify AQP ribosome-bound nascent chain complexes (RNCs) by stalling translation at defined stages in human cells and extracting directly from ER membranes using copolymer nanodiscs.

2. Compare folding trajectories across nascent chains of AQP1 and AQP4, and associated pathogenic mutants, using a range of biochemical assays.

3. Characterise nascent chain interactome and post-translational modifications of AQPs and ribosomal proteins via mass spectrometry, as well as assessing ubiquitination and UFMylation patterns linked to stress and quality control across wild-type and mutant AQPs.

4. Identify lipid species influencing folding and membrane integration using shotgun lipidomics across RNC lengths.

5. Asses the onset, functional roles and post-translational modifications as drivers of AQP4 oligomerisation.

Pellowe et al, 10.1101/2024.09.19.613857; Kitchen et al, 10.1016/j.cell.2020.03.037; Passchier et al, 10.1093/brain/awad146; Pellowe et al, 10.1021/acs.biochem.0c00423; Lu et al, doi.org/10.1091/mbc.11.9.2973

Characterising the folding environment of the human dopamine transporter

Secondary Supervisor(s): Dr Ivana Milic, Dr Alice Rothnie

University of Registration: Aston University

BBSRC Research Themes:

• Understanding the Rules of Life (Structural Biology)

Project Outline

Membrane proteins are essential for cellular function and key therapeutic targets, yet mechanistic studies of their folding and insertion during de novo synthesis on the ribosome remain limited. Intermediate states unique to directional synthesis are poorly characterised, and no method currently enables capture of folding intermediates from the human endoplasmic reticulum in sufficient quantities for advanced biophysical analysis.

hDAT is 12-transmembrane helix protein which regulates dopaminergic signalling via sodium-dependent dopamine reuptake. Misfolding and mistrafficking of hDAT contribute to Parkinson's disease, infantile dystonia, and psychiatric disorders. Patient-derived mutants often exhibit ER retention, reducing functional protein at the cell surface. This project will elucidate the factors involved in cotranslational folding and quality control of hDAT at the ER informing future therapies to correct or remove misfolded proteins to mitigate disease.

1) Capture and purify hDAT ribosome-bound nascent chain complexes (RNCs) in native nanodiscs by stalling translation at defined stages in human cells and extracting directly from ER membranes using copolymer nanodiscs.

2) Characterise nascent chain interactome and post-translational modifications of hDAT and ribosomal proteins via mass spectrometry. We will also assess ubiquitination and UFMylation patterns linked to stress and quality control across WT and mutant hDAT.

3) Identify lipid species influencing folding and membrane integration using shotgun lipidomics across RNC lengths.

4) Integrate multiomics data with in vitro translation reactions to compare folding pathways and stress responses between wild-type and mutant hDAT for real-time cotranslational folding studies.

Pellowe et al, 10.1101/2024.09.19.613857

Pellowe et al, 10.1021/acs.biochem.0c00423

Roeselova et al, 10.1016/j.molcel.2024.06.002

Srivastava et al, 10.1038/s41586-024-07739-9

Kasture et al, 10.1074/jbc.M116.737551