Imaging and reconstruction of membrane proteins with cryoEM
Time: Mon 2025-12-15 13.00
Location: Q2, Malvinas väg 10, Stockholm
Language: English
Subject area: Technology and Health
Doctoral student: Qingyang Zhang , Medicinsk avbildning, Proteinvetenskap
Opponent: Professor Peter Larsson, Linköpings universitet Institutionen för biomedicinska och kliniska vetenskaper
Supervisor: Universitetslektor Carsten Mim, Proteinteknologi
QC 2025-11-19
Abstract
Membrane proteins constitute 27% of the human proteome and play important roles in cellular processes. Dysfunction of membrane proteins causes and contributes to diverse diseases. The study of the structure and function of membrane proteins will help us to a deeply understand them. Cryo-electron microscopy (cryoEM) single-particle analysis (SPA) can provide us not only with high-resolution structures of membrane proteins but also give us the possibility to study protein’s dynamics and heterogeneous complexes they form. In this thesis, we applied SPA together with electrophysiology, surface plasmon resonance (SPR) and Microscale thermophoresis (MST) to investigate the structural and functional properties of several membrane proteins and protein complexes.
In Paper I, we studied Pannexin 1 (PANX1), an ATP release channel involved in neurological disorders and inflammation. Electrophysiology and mass spectrometry identified Y308 as a key phosphorylation site. CryoEM structures revealed that phosphorylated PANX1 and the PANX1 R75A mutant possess conformational flexibility in the transmembrane domain. Our data show that PANX1 transits between a narrow and a wide state. This transition causes the N-terminal region to be ordered (wide) or disordered (narrow). The phosphomimetic Y308E mutant only shows a wide conformation with an ordered N-terminus. Electrophysiology shows this mutation converts PANX1 into a constitutively open channel. This suggests Y308 is a phosphorylation site that locks PANX1 into the wide conformation, enabling ATP release.
Paper II presents a modified workflow for identifying and determining the structures of membrane proteins in a heterogeneous sample; without prior knowledge of the protein sequence or a model. We used a conventional suing SPA workflow to obtain cryoEM maps. These maps were modelled unsupervised with ModelAngelo and a HMMER search was done to obtain sequence information. Our data allowed us to generate three different maps from a heterogenous sample. We were able to identify each protein only based on the maps. The results were supported by mass spectrometry. The three E. coli membrane proteins were cytochrome bo3 oxidase (2.72 Å), AcrB (3.27 Å), and a previously uncharacterized E. coli ArnC protein (2.72 Å). Another important finding of this study is that MSP2N2 nanodiscs adapt their assembly around different proteins, implying that scaffold architecture depends on protein interactions.
In Paper III, we examined SMCT1 interactions with PDZK1. SMCT1 is an important membrane protein that transports monocarboxylate substrates like butyrate, lactate or niacin. It was thought that PDZK1 is an important regulator that influences the transport rate of SMCT1. We tested the affinity for SMCT1 with various assays. Our pull-down assay, SPR, and MST assays revealed only weak binding, suggesting the interaction is likely physiologically irrelevant except under conditions of local PDZK1 enrichment.
Overall, this work demonstrates the power of SPA for resolving dynamic membrane proteins, providing structural and functional insights, and establishing workflows for studying unknown or heterogeneous protein complexes.