LINXS Event - Membrane Protein workshop: Structural Resolution of Membrane Proteins: From Expression to Sample Preparation

25 May 2021, 12:30 → 26 May 2021, 12:45 Europe/Stockholm

We want to wish you welcome to the 1st Workshop from LINXS Membrane Protein Working Group. taking place digitally on Zoom.

The Membrane Protein Working Group is dedicated to explore the unique challenges involved in research on the structural biology of membrane proteins. Our first, "Structural Resolution of Membrane Proteins: From Expression to Sample Preparation'' workshop will focus specifically on three areas: expression systems, sample preparation and quality control. 

AIM

The aim is to provide expert overview about different platforms for recombinant protein productions and purification strategies for crystallography techniques. Overcoming low yield expression, misfolded proteins and difficulties in purifying stable functional proteins are among the most common bottlenecks delaying structural characterization. We invite scientists to introduce their best practices in these subjects in order to provide greater knowledge and knowledge transfer on how best to deal with these challenges.

CONTRIBUTED TALKS

We hope to engage the attendance from PhD students and postdocs from a range of disciplines, as well as more senior researchers. We are encouraging participants, and especially PhD students, to consider presenting their research in a contributed talk (call for abstracts) and also to promote lively discussions. 

CONTENT

Please find a more detailed schedule under Timetable, with abstracts from the speakers.

Day 1:

• Workshop aims and scope of the general problem

• Membrane Protein Expression
  - Comparison of different expression systems 
  - Success stories vs trouble-shooting problem areas, trial and error

• Confirmed speakers:
  Roslyn Bill (Aston University) - Recombinant membrane protein production in microbial hosts
  David Drew (Stockholm University) - An yeast GFP-based platform for functional and structures investigation of SLC transporters
  Joseph Lyons (Aarhus University) - Preparation of lipid flippases for cryo-EM studies

Day 2:

• Sample Preparation, Quality Control
  - Membrane solubilization and other preparation methods

• Confirmed speakers:
  Arjan Snijder (AstraZeneca) - Membrane protein purification and application in drug discovery
  Christopher G. Tate (Cambridge University) - Whipping GPCRs into shape for structure determination

In the autumn we will continue further on topics about membrane protein crystallization techniques, Cryo-EM or neutron structures, scattering techniques and well-diffracting crystals.

Where: Online, a zoom link will be provided just before the event
When: May 25-26 (noon-to-noon) CET 
Abstract submission deadline: 6th May 2021
Registration deadline: 20th May 2021

SPEAKERS

Roslyn Bill - Aston University, UK
Jan H Driller - Aarhus University, Denmark
Kristina Hedfalk - Gothenburg University, Sweden
David Drew - Stockholm University, Sweden
Cassie Sims - Lund University, Sweden
Julie Tucker - University of York, UK
Joseph Lyons - Aarhus University, Denmark
Charlott Stock - Aarhus University, Denmark
Arjan Snijder - AstraZeneca
Swati Aggarwal - ESS, Sweden
Vikram Dalal - Washington University in St. Louis, USA
Christopher G. Tate - Cambridge University
LineMarie Christiansen - Aarhus University, Denmark
Jörgen Ådén - Umeå University, Sweden

 

During our events we sometimes take photographs and short film clips to profile our activities. Please let us know if you don’t want to be in any photos/films before we start the event. Some talks are recorded to be used for educational purposes in the LINXS website.

By registering to our events you give your permission to LINXS, according to the General Data Protection Regulation (GDPR), to register your name and e-mail address to be used for the sole purpose of distributing newsletters and communications on LINXS activities.

Tuesday 25 May

12:30 → 12:35 Welcome and purpose of the workshop

12:35 → 13:55 Session 1 - Chair: Erika Toth

12:35 Keynote talk - Recombinant membrane protein production in microbial hosts

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have
been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of
these challenging targets. Microbial expression systems are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. A combination of rational construct design and host cell choice can dramatically improve membrane protein yields suitable for structural and functional characterization. In addition to traditional detergents, polymer-based systems are available for solubilization and biophysical characterization of these proteins, with polymers providing opportunities to investigate protein-lipid interactions.

Speaker: Roslyn Bill (Aston University, UK)

Abstract.Roslyn Bill.pdf

presentation_Roslyn_Bill.pdf

13:15 Contr. talk - The baculovirus/insect cells system

Mammalian membrane proteins are notoriously difficult to express. In contrast to bacteria or yeast, the baculovirus/insect cells system provides a good alternative to express these proteins in higher eukaryotes. Insect cells provide post-translational modifications, such as disulfide bonds or glycosylation, as well as a more akin membrane lipid composition compared to bacterial or yeast expression systems. Insect cells grow to high cell densities and using the baculovirus system even multi-protein complexes can be co-expressed.

Speaker: Jan H Driller (Aarhus University)

Abstract.membrane.Jan Driller.pdf

Jan.DrillerMembrane Protein Symposium.pdf

13:35 Contr. talk - Aquaporins – expression, purification and characterization

Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of
high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Summarizing
the production processes which has resulted in high resolution aquaporin structures reveals that Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most
commonly used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is applied after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.

Speaker: Kristina Hedfalk

abstract-K.Hedfalk.pdf

Presentation.K Hedfalk.pdf

13:55 → 14:00 Coffee Break

14:00 → 15:20 Session 2 - Chair: Susanna Horsefield

14:00 Keynote talk - An yeast GFP-based platform for functional and structures investigation of SLC transporters

Recombinant expression screening of eukaryotic membrane proteins for functional and structural investigation is still a trial-and-error process. We have found that cloning gene-strings by homologuous recombination into a 2u vector S. cerevisiae expression vector and then detecting membrane protein expression and stability by working with GFP-fusions, enables many constructs to be rapidly tested. We have found an excellent correlation between the best-behaving membrane proteins in yeast to those produced by transient transfection in mammalian HEK293 cells. Here, I will outline these GFP-based methods with a focus on ion and sugar SLC transporters.

Speaker: David Drew (Stockholm University, Sweden)

abstract.David Drew.pdf

14:40 Contr. talk - Study of anti-cancer effects of TTA-A2 and paclitaxel due to antagonistic interactions with T-type calcium channels

Studies have shown that in cancer cells, there is an increased T-type calcium channel (TTCC) expression compared to healthy cells. Therefore, the studies targeting TTCC for cancer therapy
have shown many positive outcomes. Here, we have used TTA-A2- a potent TTCC inhibitor as a test drug, and paclitaxel (PTX)- a tubule-binding anti-cancer agent as a positive control. Blocking
TTCC has shown to overcome resistance in cancer cells towards anti-cancer drugs by reducing calcium influx, and some studies have shown that PTX treatment also reduces the intracellular calcium signaling in cells. So, there is a possibility that PTX might be interacting with calcium channels. Since, drug-drug interaction can cause severe side-effects, or alter the actions of each other; we aim to study the interactions among TTA-A2, PTX, and TTCC. Therefore, in this study we have analyzed the binding of of TTA-A2 and PTX with TTCC. Our results showed that both the drugs, TTA-A2 and PTX, could interact at the same site of TTCC to form a higher stable complex as compared to the TTCC-native. The result indicated that sequential treatment could help to overcome the antagonistic interaction between the two drugs.

Speaker: Vikram Dalal (Washington University in St. Louis)

abstract-V.Dalal.pdf

Vikram_Dalal_ppt.pdf

15:00 Contr. talk - Production of insect odorant receptors in yeast for structural studies

Insect olfactory receptors (ORs) are proteins involved in olfaction in insects. Insect ORs are 7-transmembrane domain proteins that possess a unique structure, forming a heteromeric complex with a highly conserved co-receptor (ORCO). The heteromeric complex constitutes an ion channel, which opens allowing movement of ions upon activation by a ligand. Specificity of ORs is variable, and little is understood about where this specificity arises and how the binding of an active ligand subsequently opens the ion channel. Despite significant functional studies of insect ORs, only two structures, ORCO
from the fig wasp Apocrypta bakeri, and a homomeric complex of OR5 from the jumping bristletail, Machilis hrabei, have been determined. This structural work relied on a well-established
expression system, HEK293 cells, which are commonly used for functional screening of ORs. However, HEK cells are not an optimal system for protein production for structural analysis. In this project, we begin to develop a more efficient protein production system for structural investigation of insect ORORCO
complexes, focusing on ORs of Lepidoptera and Culicidae (moths and mosquitoes). Initial work has focused on the expression of insect OR-ORCO complexes with a fusion eGFP in a Pichia pastoris yeast.

Speaker: Cassie Sims (Lund University)

Abstract.Cassie Sims.pdf

15:20 → 15:30 Coffee Break

15:30 → 16:30 Session 3 - Chair: Maria Gourdon

15:30 Keynote talk - Preparation of lipid flippases for cryo-EM studies

P4-ATPases are lipid flippases that drive the ATP-dependent inward translocation (flipping) of lipids within the membrane. They are members of the P-type ATPase superfamily and largely function as binary complexes with an auxiliary protein from the CDC50 family. Select lipid flippases are auto-regulated by conserved motifs in their termini and require activation by an external stimulus, i.e., binding of an activating lipid or regulatory protein, and/or specific phosphorylation of the termini. To elucidate the auto-regulation and transport mechanisms, we determined the structures of yeast and mammalian lipid flippases by cryo-electron microscopy.

In this talk, I will give an overview of the tour de force we embarked on, from expression through purification to cryo-EM imaging of these lipid flippases.

Speaker: Joseph Lyons (Aarhus University, Denmark)

abstract_joe_lyons.pdf

16:10 Contr. talk - Production and purification of the K+-dependent P-Type ATPase KdpFABC for crystallization and cryo-EM

At very low extracellular K+ concentrations (<100 μM), KdpFABC rescues bacterial cells by taking up K+ against an up to 105-fold concentration gradient. To understand the mechanism of this heterotetrameric complex, KdpFABC was
purified for functional and structural investigations. A new expression system for KdpFABC was generated, a 3-step chromatographic purification implemented and protein functionality in different detergents as well as in the presence of
inhibitors was tested. The work was performed in the lab of Prof. Inga Hänelt at the University of Frankfurt from 2014-2020.

Speaker: Charlott Stock (Aarhus University)

abstract.C.Stock.pdf

16:30 → 16:35 Short Break

16:35 → 17:00 General Discussion (Chair: Susanna Horsefield)

Wednesday 26 May

09:00 → 10:20 Session 4 - Chair: Karin Lindkvist

09:00 Keynote talk - Membrane protein purification and application in drug discovery

In the presentation I will discuss our capabilities, processes and methods for membrane protein expression and purification, including our recently developed teabag purification method. The teabag method allows rapid purification of membrane proteins with reduced hands-on and processing time. Application of membrane proteins in drug discovery will be illustrated by our work on the protease-activated receptor 2, where extensive protein engineering allowed DNA-encoded library screening on purified receptor, establishment of biophysical assays, and structure assisted drug design.

Speaker: Arjan Snijder (Astra Zeneca)

Abstract.Arjan Snijder.pdf

Arjan Snijder Membrane Protein Symposium May 2021.pdf

09:40 Contr. talk - Towards Neutron crystallography of membrane proteins: Insights into production of deuterium-labelled OmpF

Hydrogen bonds play a crucial role for protein function and involved in almost every mechanism from foundation of protein structure to enzyme catalysis. Hydrogen (1H) atoms form the basis of hydrogen bond that is not scattered by X-ray crystallography due to its poor scattering power. Neutron protein crystallography (NPX) is a powerful tool that is capable of locating hydrogens and study the significance of hydrogen bonding interactions in biomacromolecules. However, due to
the requirement of large crystals very few neutron structures have been deposited in PDB with no membrane protein structure determined yet. Additionally, 1H has a negative scattering length and large incoherent cross-section giving rise to a significant background noise in neutron data collection.
This effect can be minimized by isotopic substitution of 1H with its heavier isotope deuterium (2H or D) leading to less ambiguous data analysis and better structure interpretation. Overall ˜25% H atoms in a protein are solvent exchangeable and can be exchanged by dissolving in heavy water. However, complete deuterium labelling (perdeuteration) is required for the remaining 75% H atoms. In this work, an optimized methodology for large scale production of perdeuterated bacterial outer membrane protein F (OmpF) has been designed. OmpF was produced in deuterated minimal medium with different carbon sources. Mass spectrometry and thermal stability experiments
verified the purity and level of deuteration of OmpF protein. Perdeuterated OmpF crystals also diffracted X-rays to 9 Å resolution emphasising the need of fine tuning of perdeuterated crystallisation conditions.

Speaker: Swati Aggarwal (European Spallation Source)

abstract-S.Aggarwal.pdf

SwatiAggarwal_LINX_2021.pdf

10:00 Contr. talk - Production of Bcl-2 proteins involved in regulation of mitochondrial apoptosis

Programmed cell death (apoptosis) is essential for human life. In its intrinsic pathway, the Bcl-2 (B-cell lymphoma 2) protein family regulates cell life and death by controlling permeability of the mitochondrial outer membrane (MOM). However, the molecular basis of cell protection by its anti-apoptotic Bcl-2 members remains elusive due to the lack of detailed structural insight into their action at the MOM to ensure its integrity. To provide atomic-level insight into their functioning, we will use the founding member of this family, the human antiapoptotic Bcl-2 protein itself, whose involvment in p53 regulation and its overexpression
plays a notorious role in many cancers and their treatment resistance. However, for a long time, obtaining sufficient protein was cumbersome due to its insolubility as a membrane
protein and low yields. Therefore, we establishes an expression and purification protocol [1] to produce routinely mg amounts of the fully functional full-length human isoform 2 of Bcl-2
(Bcl-2(2)) which can also be isotopically labelled as 15N/13C/2H versions, ideally suited for neutron and NMR studies. The protocol even allows to generate residue specific Bcl-2
mutants and various constructs [2].

1. Aden, A.U. Mushtaq, A. Dingeldein, M. Wallgren, G. Gröbner. A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2. Protein expression and purification. 172 (2020) 105628.
2. A. Ul Mushtaq, J. Aden, T. Sparrman, M. Hedenstrom, G. Gröbner. Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promising Cancer Drug Target. Molecules 26 (2021) 1467, 1-14

Speaker: Jörgen Ådén (Umeå University)

Abstract_Lund_Gröbner.Åden_2021.pdf

Lund_2021_Jörgen Åden.pdf

10:20 → 10:30 Coffee Break

10:30 → 11:50 Session 5 - Chair: Swati Aggarwal

10:30 Keynote talk - Whipping GPCRs into shape for structure determination

Over the past 15 years we have determined multiple structures of GPCRs by both X-ray crystallography and cryo-EM. For each GPCR we had to overcome the difficulties of overexpression, solubilisation, stabilisation and structure determination, as you would for any membrane protein. For X-ray crystallography, extensive protein engineering was required to form well-diffracting crystals, in parallel with judicious choices of detergent, ligand and crystallisation format (vapour diffusion or lipidic cubic phase). Different strategies also had to be devised depending on which conformational state was required for structure determination. The advent of high-resolution structure determination by single particle cryo-EM has presented new opportunities for GPCR structural biology, and structures are now being determined that would have been onerous, if not impossible, to determine by X-ray crystallography. I will present the key factors for expression, purification and structure
determination that have made GPCR structures possible.

Speaker: Christopher G. Tate (MRC Laboratory of Molecular Biology, UK)

Abstract.Christopher G Tate.pdf

Presentation.C Tate.pdf

11:10 Contr. talk - From expression to purified protein: a P4-ATPase for CryoEM studies

The Saccharomyces cerevisiae P4-ATPase Neo1p is believed to function as a lipid flippase, translocating lipids towards the cytosolic leaflet of both Golgi and endosome membranes. In
order to screen around both functional and structural studies to ascertain the exact role and function of Neo1p an efficient high-yield expression and purification protocol is vital. By
overexpression in S. cerevisiae and purification based on a high-specificity tag we are left with a pure protein sample ideal for both structural and functional studies. For CryoEM
detergent-exchange into LMNG can be easily performed, with some attention to detergent concentration

Speaker: LineMarie Christiansen (Aarhus University)

abstract.LM Christiansen.pdf

11:30 Contr. talk - Small-scale expression and purification of the cytokine receptor, MPL, using the ALiCE® cell-free system

MPL (also called TpoR) is the receptor for the haematopoietic cytokine, thrombopoietin (TPO). Together, MPL and TPO control the production of platelets and the maintenance of haematopoietic stem cells. Gain of function mutations in MPL constitute ˜5-10 % of driver mutations in essential
thrombocythemia and primary myelofibrosis, whilst loss of function mutations give rise to thrombocytopenias. MPL comprises an ˜500 aa extracellular domain, a single transmembrane helix, and an ˜120 aa intracellular domain. The extracellular domain contains the thrombopoietin binding site, whilst the intracellular domain provides the binding site for Janus kinase 2, as well as other downstream signalling molecules, including the Signal Transducers and Activators of Transcription
(STAT) proteins. Structural information on MPL is lacking, in part due to the challenges of producing recombinant protein. Endogenous expression levels of MPL are very low (< 5000
receptors/cell) and heterologous expression of full-length MPL has not been reported. Here we report small-scale expression and purification of both full-length and ectodomain of MPL using the ALiCE® cell-free expression system (LenioBio GmbH). This plant cell-based system allows for the expression of membrane and secreted proteins within the microsomal compartment, ensuring their correct folding, membrane insertion and glycosylation. Optimisation of expression time, plasmid
concentration and downstream processing allowed for production and purification of glycosylated MPL, the functional characterisation of which is ongoing.

Speaker: Julie Tucker (University of York)

abstract-J.Tucker.pdf

Julie.Tucker_LINXS_workshop.presentation.pdf

11:50 → 11:55 Short Break

11:55 → 12:20 General Discussion (Chair: Urban Johanson)

12:20 → 12:25 Closing remarks, introducing the autumn workshop