PI: Emma Sparr

Name of project:
Membrane-induced liquid-liquid phase separation

Membrane-induced liquid-liquid phase separation (MI-LLPS) of proteins is a hypothetical process, where cellularly dissolved proteins interact with lipid membranes to form liquid microdroplets.

I want to show experimentally, that proteins undergo MI-LLPS and link this process to molecular properties of the respective molecules (proteins and lipids). While LLPS is a relevant process for the physiological functions of the proteins, by maturation and rigidification these liquid microdroplets can also transition into cytotoxic oligomers and amyloid fibrils, which are directly linked to the pathogenesis of diseases like Parkinson or Alzheimer.

Therefore, I will study model proteins with shown LLPS in the presence of lipid model membranes to observe under which experimental conditions membrane-induced LLPS takes place. In the first part different model systems with varying protein concentrations with and without presence of a membrane (+crowding agent) will be investigated using fluorescent labelled protein and confocal microscopy.

In the second part I will characterize the process in-depth using a combination of QCM-D and NMR-techniques.

Main objectives and aims of the project

• Observe liquid-liquid phase separation that is induced e.g nucleated by lipid membranes

• Characterize the interactions between lipids and membrane as well as of the liquid protein microdroplets

• ·Link these observations to physiological and pathological effectsDescription text goes here

PI: Emma Sparr

Name of project:
Impact of Nanoparticle Binding on Lipid Redistribution and Membrane Deformation in Cellular Membranes

PI: Karin Tran Lundmark

Name of project:
3D imaging of pulmonary vascular remodelingon

Pulmonary fibrosis (PF) is a progressive, severe, and often lethal pulmonary disease. End-stage disease is characterized by extensive deposition of extracellular matrix (ECM), leading to parenchymal scarring and advanced pulmonary vascular remodeling, which can lead to heart failure. Current treatments can slow disease progression but do not resolve tissue remodeling. Most PF studies have been conducted at the end stage of the disease, which limits understanding of its progression. Additionally, the complex anatomical structure of the lung and the spatial and temporal heterogeneity of the fibrotic process make it difficult to understand the disease using traditional two-dimensional (2D) histological methods. Therefore, comprehensive three-dimensional (3D) studies are needed.

This project aims to use synchrotron-based phase-contrast micro-CT imaging to study the 3D intricacies of PF pathogenesis in both human lung tissue and animal models, spanning from early to end-stage disease. By combining micro-CT with conventional histology, immunohistochemistry, multi-color fluorescence immunostaining, spatial transcriptomics, and in situ hybridization, the project aims to identify the contribution of ECM to the causal and longitudinal relationship between parenchymal and vascular remodeling during disease progression. The multifaceted analysis also aims to identify microanatomical changes in the pulmonary vasculature correlated with adverse clinical outcomes, potentially revealing biomarkers for further patient stratification.

By unraveling the 3D dynamics of vascular abnormalities and parenchymal remodeling across disease stages and animal models, this project lays the groundwork for identifying new targets for novel therapeutic interventions for PF patients. .

PI: Martin Bech, Filip Szczepankiewicz

Name of project:
Pre-clinical exploration of prostate cancer microstructure by MRI and micro-CT

Prostate cancer is deadly disease which causes the largest number of cancers related deaths in men. In a clinical setting, the most promising imaging method is arguably diffusion magnetic resonance imaging (dMRI) that enables non-invasive tissue characterization. But their spatial resolution is relatively low and the fundamental contrast mechanisms of microstructure MRI have yet to be fully understood. To resolve this gap, synchrotron-based phase contrast computer tomography can be used to depict prostate tissue architecture across multiple scales.

Simultaneously, trace elements serve as critical regulators of prostatic pathophysiology; the human prostate accumulates zinc (Zn) at concentrations 2–5 times higher than other tissues, whereas prostate cancer tissue exhibits a marked depletion of Zn and iron (Fe). X-ray fluorescence (XRF) offers the ability to map the spatial distribution of these key trace elements. Since dMRI parameters are highly sensitive to heterogeneity and structural configuration, we hypothesize they will correlate with analogous features quantified by µCT and XRF. By mapping these associations, we aim to elucidate the biophysical origins of the observed MR signal, thereby advancing the interpretation of current imaging protocols and the design of future MRI methods.

Main objectives and aims:

We aim to establish whether MRI can serve as an accurate marker for prostate cancer by investigating the correlations between dMRI, microvasculature derived from µCT, and tissue immunohistochemistry. These results hold the potential to enhance diagnostic specificity and pave the way for MRI-driven “virtual biopsies” in oncology.

PI: Felix Roosen-Runge

Name of project:
Microscopic structure in plant seeds by scanning X-ray diffraction

Proteins from plant seeds present a promising alternative to animal-derived food proteins as a more sustainable, healthy and affordable protein source. However, plant seeds store macromolecules in so-called protein bodies, which evolved to preserve protein functionality over a long time, providing a stable energy and nutrient source for the next plant generation during seed germination. The disintegration of food proteins from these highly stable structures in an energy and resource-efficient way is an ongoing challenge, as typical extraction methods use harsh solvents and elevated temperatures.

The objective of this project is to characterize the microscopic structure in plant seeds and its evolution during processing. To this end, microfocus scanning small and wide-angle X-ray scattering (SWAXS) and X-ray fluorescence (XRF) will be applied to typical plant seeds used for food applications, including, among others, pea, soy and wheat. Using a combined analysis of SWAXS and XRF, the results will not only provide fundamental insights into colocalization of macromolecular and ionic constituents but also pave the way towards more efficient and predictive protocols for food applications.

Studies will be extended to include plant-based food proteins at various processing steps to optimize sustainable processing and production of plant protein-based food.

Main objectives and aims

• Detailed characterization of the molecular packing structure within plant seeds, providing fundamental insights into plant protein and seed structure

• Link between elemental composition, local ordering and aggregation

• Connection between molecular and mesostructural features

• Observation of microstructural evolution during food processing to support more predictive protocols for food applications

PI: Martin Bech

Name of project:
X-ray fluorescence mapping of biological tissue

Recent advances in synchrotron X-ray imaging have made X-ray fluorescence (XRF) a powerful tool for visualizing how chemical elements are distributed within biological tissues. These elemental patterns play an important role in many diseases, yet they remain difficult to study at the relevant length scales.

This project aims to develop an advanced synchrotron-based imaging workflow that combines XRF with complementary techniques. Together these methods will allow biological tissues to be examined across multiple length scales, from overall morphology down to nanoscale structural and chemical features.

As a demonstration system, carotid arterial tissue affected by atherosclerosis will be used. By correlating elemental composition with nanoscale structural information, the project seeks to improve our understanding of plaque composition and stability—key factors in cardiovascular disease and stroke risk.

The research will be carried out at beamlines with complementary capabilities. Clinical and laboratory-based X-ray imaging, as well as histology, will guide the selection of regions of interest.

Beyond atherosclerosis, the developed methodology will be broadly applicable to other biological tissues, including kidney and cancer samples. Overall, the project will deliver a flexible and transferable imaging approach that supports interdisciplinary research and contributes to improved understanding of disease mechanisms with potential relevance for clinical research and future therapies.

Main Objectives and Aims:

• Develop a flexible multimodal imaging workflow combining XRF and other complementary synchrotron-based techniques 

• Enable detailed mapping of elemental composition and nanoscale structure in biological tissues

• Apply the workflow to study carotid arterial tissue and atherosclerotic plaques, link elemental distributions to structural features relevant for plaque stability and rupture

• Integrate synchrotron imaging with clinical CT, micro-CT, and histology

• Optimize data acquisition, processing and analysis methods

• Demonstrate the broader applicability of the approach to other tissue types, such as kidney and cancer samples

Shuvasis das Gupta
Lund University
PI: Hanna Isaksson

Name of project:
High resolution imaging of the bone-cartilage interface in the knee during osteoarthritis

PI: Pontus Gourdon

Name of project:
Development of ion channel activators and inhibitors

The five KCNQ (Kv7 or M-type) voltage-gated potassium channels (KCNQ1–5) are essential in maintaining neuronal excitability and cardiac rhythm. Gain or loss of function mutations in these channels are implicated in a palette of neurological and cardiovascular disorders, including epilepsy and Long QT syndrome (LQTS). As a result, considerable academic and translational efforts have focused on identifying small-molecule modulators of KCNQ activity. Among these, polyunsaturated fatty acids and mimics thereof (PUFAs) have emerged as promising candidates, capable of modulating the activity of all five KCNQ channels and rescuing defective KCNQ1 function in LQTS. However, the modulatory effects of PUFAs cannot be predicted and are sometimes subtype-specific, and the underlying mechanisms remain poorly understood, which limits both our understanding and application of KCNQ modulators. In this project, through combined structural and functional analyses, we aim to uncover the molecular determinants of PUFA on the KCNQs. These insights will provide a foundation for rational drug design strategies and advance the therapeutic potential of PUFAs in treating KCNQ-related diseases.

Aims

1.   Overproduction and purification of KCNQ subtypes. Protein expression will be performed using the HEK cells expression system.

2.   The function of PUFA/PUFAs on KCNQ channels. Electrophysiological analyses of PUFA-mediated modulation of KCNQ subtypes will be performed.

3.   Structural characterization of the molecular effect of PUFAs on KCNQ channel. Cryo-EM particle screening and data collection will be performed at the local cryo-EM facility located at MAX-IV in Lund, and at other Scandinavian state-of-the-art microscopes.

PI: Agnieszka Chacinska and Remigiusz Serwa

Name of project:
Functional alterations of protein import into mitochondria

Mitochondria are essential cellular organelles responsible for energy production and metabolic regulation. Although they contain their own genome, the vast majority of mitochondrial proteins are synthesized in the cytosol and imported into the organelle via dedicated transport machineries. The TIM23 complex is a central component of this import pathway, mediating the translocation of presequence-carrying proteins into the mitochondrial matrix and the inner mitochondrial membrane. The core of this complex is formed by the half-channel-forming proteins TIM23 and TIM17. They are oriented back-to-back, with TIM17 containing the translocase function. In yeast, the TIM23 complex has been studied in detail, but its human counterpart remains poorly understood. Interestingly, human mitochondria contain two paralogous TIM17 proteins, TIMM17A and TIMM17B, which form distinct TIM23 complexes that contain either paralogue. While TIM17B is the housekeeping component, the TIM17A-containing complex is subject to high turnover.

This project aims to understand the assembly and biogenesis of different TIM23 complexes in mammalian cells. Using classical in organello import assays and newly established in vivo protein labeling, in conjunction with native PAGE, will help elucidate the dynamics of TIM23 complex composition. Studying the regulation of these complexes under stress will further elucidate how they selectively regulate mitochondrial protein import. In the long term, these insights will define new principles of mitochondrial quality control and may reveal novel strategies for addressing mitochondrial dysfunctions underlying human disease.

Main objectives and aims:

• Staging  of mammalian TIM23 complex assembly

• Identification of regulatory factors involved in TIM23 complex biogenesis

• Elucidate regulatory mechanisms controlling degradation and stress responsiveness

• Define how distinct TIM23 complexes contribute to mitochondrial homeostasis and adaptation

PI: Frank Gabel

Name of project:
Developing new tools for structural biology of biomacromolecular complexes in solution: a joint SAXS/SANS contrast variation study of the membrane protein FhuA

Membrane proteins and other bio macromolecular assemblies are central to biology and biomedicine, yet their internal organization in solution is often difficult to resolve because they are dynamic, heterogeneous, and composed of multiple components (protein, lipids, nucleic acids, detergents/nanodiscs). This project will develop and validate a joint SAXS/SANS contrast-variation strategy to obtain unprecedented structural information on such complexes in solution.

Small-angle X-ray scattering (SAXS) provides high-throughput measurements sensitive to electron density, while small-angle neutron scattering (SANS) offers unique access to contrast variation through H2O/D2O exchange and selective deuteration, enabling specific components to be highlighted or “masked”. Building on recent advances in solvent-contrast variation SAXS (using electron-rich contrast agents) and established SANS contrast-variation methods, we will perform an extensive, coordinated SAXS/SANS study of the solubilized membrane protein FhuA as a model system, including tailored contrasts for proteins, lipids, and nanodiscs.

The outcome will be an integrated experimental and data-analysis workflow that exploits the unusually broad and complementary contrast space of X-rays and neutrons to separate contributions of different components and to refine solution-state structural models. Once validated on FhuA, the approach will be transferable to a wide range of challenging bio macromolecular complexes relevant to structural biology and biomedical research.

Main objectives and aims:

• Establish an integrated SAXS/SANS contrast-variation workflow for multicomponent bio macromolecular complexes in solution.

• Implement solvent-contrast variation SAXS (electron-rich contrast agents) and SANS H2O/D2O contrast variation in a coordinated experimental design.

• Apply selective deuteration strategies (protein and/or lipid/nanodisc) to enable component-specific visibility in SANS.

• Use FhuA membrane protein complexes as a benchmark system to validate the methodology and quantify achievable component separation.

• Develop and apply robust data reduction, modeling, and joint-fitting approaches that combine SAXS and SANS across multiple contrasts.

• Extract structural parameters describing overall shape, internal organization, and component arrangement (protein vs. lipid/nanodisc) in solution.

• Deliver transferable protocols, analysis guidelines, and reference datasets enabling broader adoption for other membrane proteins and bio macromolecular assemblies.

PI: Agnieszka Chacinska

Name of project:
TIM23 Complex Regulation during Mitochondrial Protein Import

Mitochondria are essential metabolic hubs that depend on the efficient import of nuclear encoded proteins to maintain cellular health. Central to this process is the TIM23 translocase, a multisubunit molecular machine that facilitates protein translocation across the inner mitochondrial membrane. This research investigates the mechanistic factors influencing protein import, with a specific focus on the core subunits TIMM50, TIMM17A, and TIMM17B. While TIMM50 serves as the critical gatekeeper for protein recognition, the paralogs TIMM17A and TIMM17B are thought to provide a regulatory switch for the import channel. However, how these specific components interact to maintain structural integrity or drive complex disassembly during cellular stress remains largely unknown. By employing an integrative suite of biochemical assays and molecular tools, this study explores the signaling cascades and structural remodeling required to adapt the TIM23 apparatus to proteostatic stress. Our research investigates the pathways that safeguard mitochondrial biogenesis during periods of instability, specifically characterizing how subunit composition is remodeled to prevent metabolic collapse. These findings offer critical insights into the molecular pathologies associated with import failure and provide a framework for understanding mitochondrial resilience in the face of cellular stress.

Main Objectives and Aims:

• To investigate how subunit assembly shift under various stress conditions.

• To identify the pathways that communicate cellular distress to the mitochondria.

• To determine the cellular machinery responsible for the targeted breakdown of specific translocase components during mitochondrial remodeling.

PI: Karin Lindkvist

Name of project:
Targeting aquaporins for the development of novel therapeutic and pest control strategies

Aquaporins (AQPs) are conserved integral membrane proteins critical for transporting water and small solutes across cell membranes, playing key roles in cellular signalling and homeostasis in both mammals and insects. This project explores AQPs as a promising target for human therapeutics and sustainable pest control. In cancer, AQP-mediated hydrogen peroxide (H₂O₂) transport contributes to redox adaptation in T-cell acute lymphoblastic leukemia (T-ALL) cells, enabling resistance to chemotherapy-induced oxidative stress and apoptosis. Inhibiting AQP could therefore sensitize these cells to treatment, offering a novel strategy to improve outcomes for T-ALL patients. In agriculture, AQPs are crucial for water regulation in beetle pests like the red flour beetle Tribolium castaneum, which face high desiccation risk due to their small size. Exploiting species-specific differences in insect AQPs structures opens the door to developing insecticides with minimal off-target effects on non-pest organisms and the environment.

To achieve the above, several key AQPs will be expressed and purified for single-particle cryo-electron microscopy (cryo-EM) analysis, and the resulting high-resolution structures will be combined with computational drug design to create potent, isoform-selective inhibitors. Candidate compounds will be validated through functional assays in relevant cell and insect models. Anticipated outcomes include confirming AQP's viability as a therapeutic/pest control target, alongside structural data to guide the rational design of next-generation AQP inhibitors for both medical and agricultural applications.

Aims and objectives:

Determine high-resolution structures of key AQPs to facilitate rational drug design.

a. Purify AQP proteins expressed in yeast cells (P. pastoris) and determine optimal conditions for cryo-EM grid preparation (e.g., reconstitution in lipid nanodiscs).

b. Collect and process single-particle cryo-EM data of selected AQP samples to obtain high-resolution structures.

c. AQP structures will be used to inform the design of compounds blocking their activity using computational methods in an iterative manner.

Assess the viability of targeting AQPs in the development of eco-friendly insectides for the beetle pest Tribolium castaneum.

a. Candidate Tribolium castaneum AQPs (TcAQPs), such as TcEglp3, TcEglp4 and TcDrip, will be assessed for substrate selectively using swell-based uptake assays.

b. Compounds from Aim 1 will be assessed on their ability to block TcAQP-mediated solute transport with organ assays (e.g., fluid secretion assays) from natively-dissected osmoregulatory tissues.

c. Compounds shown to be effective in blocking TcAQP activity ex vivo will be tested for their in vivo impact on water retention and desiccation tolerance in Tribolium castaneumbeetles. In addition, ecotoxicological safety testing on beneficial insect species such as Coccinella septempunctata and human cell lines will be performed.

PI: Pontus Gordon

Name of project:
Basic and applied science targeting P-type ATPases

Copper homeostasis in human cells is maintained by two closely related P-type ATPases, ATP7A and ATP7B, which transport Cu⁺ ions across cellular membranes. Dysfunction of these transporters leads to the severe Menkes and Wilson’s disease, respectively. Despite recent progress in structural studies, the molecular mechanism by which copper is transported during the catalytic cycle remains poorly understood. This project aims to reveal how copper is transferred from the cytosolic chaperone ATOX1 to the human copper-transporting P-type ATPases and how this process is coupled to active transport across the membrane. By combining biochemical and biophysical approaches with advanced cryo-electron microscopy, including methods capable of capturing transient intermediate states, the project seeks to visualize key steps of copper transfer in action. Together, these studies will provide a detailed mechanistic view of copper transport by human P-type ATPases and establish a structural framework for understanding how copper transfer is coordinated at the molecular level.

Aims and objectives:

• Dissect the molecular mechanism of copper delivery to ATP7A/B

• Understand regulatory roles of metal-binding domains in copper transport

• Resolve structural features of the high-affinity copper-binding site

• Reveal intermediate states of the ATP7A/B transport cycle using cryo-EM

PI: Mikael Lund

Name of project:
Modelling solution thermodynamics and scattering in complex biomolecular samples

Protein-protein interactions (PPIs) have attracted significant attention because of their pivotal role in life. For example, many human diseases are associated with aberrant PPIs. Meanwhile, PPIs have emerged as a promising therapeutic approach for the pharmaceutical industry. Over the past decades, it has been shown that hydrophobic interactions (HI) are one of the most important contributors to PPIs, strongly affecting the solution behavior and thermodynamic stability of proteins. HI involve enthalpic and entropic contributions and exhibit nontrivial temperature dependence. Although the exact molecular basis for these contributions is still under study, numerous theoretical models have been developed for studying PPIs while accounting for the temperature dependence of HI.

However, most of these models were originally designed for intrinsically disordered proteins (IDPs), which considerably reduces their accuracy when applied to globular proteins. This project aims to develop a highly realistic coarse-grained model for globular proteins by adapting the temperature-dependent approaches for HI derived for IDPs. While our primary focus is on understanding the effect of temperature on HI, other important contributions to PPIs, such as electrostatic interactions, will also be taken into account.

Consequently, we will study PPIs under a range of solution conditions, including variations in temperature, pH, ionic strength, and protein concentration. Finally, we will validate the proposed model by comparing the second virial coefficient, B2 obtained from calculations of the potential of mean force (PMF), and the structure factor, S(q), obtained from Monte Carlo simulations, with corresponding experimental data from SLS and SAXS.

General objective:

To develop a highly realistic coarse-grained model of globular proteins that accounts for the temperature dependence of protein-protein interactions, focusing on hydrophobic interactions, using a combination of two-body potential of mean force calculations, many-body molecular simulations, and advanced scattering techniques.

Specific objectives:

• To investigate and compare different temperature-dependent approaches for modeling PPIs in coarse-grained models of proteins, with an emphasis on hydrophobic interactions.

• To implement the temperature dependence of hydrophobic interactions in a highly realistic coarse-grained (CG) model of globular proteins.

• To perform a series of two-body potential of mean force calculations (PMF) in the software Duello and many-body Metropolis Monte Carlo (MC) simulations in the software Faunus, using the augmented model.

• To calculate key physicochemical properties of protein solutions using the new CG model under varying conditions of temperature, pH, ionic strength, and protein concentration. This includes computing the second virial coefficient, B2 from two-body calculations of PMF, and the structure factor S(q), from many-body Metropolis MC simulations.

• To validate the CG model and the computed properties by comparing them with corresponding experimental data obtained from static light scattering (SLS) and small-angle X-ray scattering (SAXS).

PI: Vasili Hauryliuk

Name of project:
Molecular mechanisms of antiphage defense

Phages are antimicrobial agents capable of specifically recognizing and killing bacteria, making phage therapy a promising co-adjuvant for treating multi-drug-resistant bacterial infections. However, bacteria have evolved a wide range of anti-phage defence systems to counter phage infection.

To successfully apply phages for therapeutic purposes, a deeper understanding of anti-phage defence systems is essential. The bacterial membrane represents the first point of contact during phage infection and is therefore a critical site for detecting and counteracting invading phages. Despite this, most currently characterized defence systems are cytosolic, leaving membrane-associated systems largely unexplored.

In this project, we aim to leverage topology-based bioinformatic approaches to identify novel membrane-associated defence systems. These systems will then be structurally characterized using single-particle cryo-electron microscopy (cryo-EM) to elucidate their molecular interactions and activation mechanisms. Finally, we will apply structure-guided protein design to dissect their mechanisms of action and develop inhibitors capable of overcoming these defence systems, thereby enabling productive phage infection.

This research will advance our understanding of phage–host interactions and provide new strategies to enhance the efficacy of phage therapy against antibiotic-resistant bacteria.

Main objectives and aims:

• Identification of novel membrane-associated anti-phage defence systems

• Structural and mechanistic characterization of these systems

• Design of inhibitors targeting anti-phage defence systems to enable phage infection

   

PI: Trevor Forsyth

Name of project:
Understanding and optimizing mRNA therapeutics: structural characterization of m-RNA lipid-based nanoparticles (LNPs) by X-ray and neutron small-angle scattering and cryo-electron microscopy

This research project aims to advance the design of next-generation vaccines for infectious diseases and cancer immunotherapy. The primary focus is the detailed structural characterization of lipid nanoparticle (LNP) systems, which are critical carriers for mRNA delivery.

By utilizing a multiscale approach that combines synchrotron small-angle X-ray scattering (SAXS), neutron scattering (SANS), and cryo-electron microscopy (cryo-EM), the project seeks to elucidate the complex internal organization of these nanoparticles. The goal is to generate high-quality structural data that reveals the essential structure-activity relationships (SAR) governing LNP performance. Conducted in collaboration with major partners including CureVac (BioNTech), the Institut Laue-Langevin (ILL), and the European Spallation Source (ESS), this work integrates academic innovation with industrial requirements to accelerate the development of more efficient and stable mRNA vaccine formulations.

Main Objectives and Aims

• Elucidate Structure-Activity Relationships: Generate high-resolution structural data to determine how the internal organization of lipid nanoparticles influences their biological activity and efficacy, directly informing the design of improved vaccine formulations.

• Apply Advanced Characterization Techniques: Design and conduct comprehensive structural studies utilizing a combination of synchrotron radiation, neutron scattering techniques, and cryo-electron microscopy to resolve LNP morphology at multiple scales.

• Advance Next-Generation Therapeutics: Lead research focused on optimizing delivery systems for specific applications in infectious disease prevention and cancer immunotherapy.

Bridge Academic and Industrial Research: Integrate foundational academic research with industrial objectives through collaboration with partners like CureVac (BioNTech) and large-scale facilities (ILL, ESS), ensuring research findings are translatable to real-world pharmaceutical development.

PIs: Yolanda Markaki, Cyril Dominguez and John Schwabe

Name of project:
Protein structure in health and pathology

PIs: Hanna Kwon, Andrew Hudson and Peter Moody

Name of project:
Protein structure in health and pathology

Haem is an indispensable iron-containing cofactor essential for numerous biological processes. It serves as a prosthetic group in haemoproteins such as globins, cytochromes, and peroxidases, enabling oxygen transport, electron transfer, and catalysis, while also functioning independently as a regulatory and signalling molecule, including in the modulation of circadian rhythms. Free haem is cytotoxic due to its ability to generate free radicals through Fenton reactions. Consequently, the majority of cellular haem remains tightly bound to proteins, with a small fraction forming a weakly bound, exchangeable haem pool. Proteins that bind and deliver this exchangeable haem to specific target proteins are known as haem chaperones. Recent studies indicate that proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and progesterone receptor membrane component 1/2 (PGRMC1/2) contribute to intracellular haem trafficking, including from mitochondria to other cellular compartments. Moreover, haem serves as an endogenous ligand for nuclear receptors such as REV-ERBα/β, CLOCK and PERIOD which are integral components of the mammalian circadian clock. Binding of haem to these transcription factors enhances their repressive activity on core clock genes, including BMAL1, thereby linking intracellular haem availability directly to rhythmic gene expression and metabolic homeostasis.

However, the molecular details of haem exchange and binding mechanisms remain elusive. Insights into these processes may clarify the regulatory roles of haem and its chaperones in circadian rhythms, as well as broader signalling functions. Ultimately, findings could advance understanding of fundamental biological processes and inform therapeutic strategies for disorders linked to haem homeostasis dysregulation, such as porphyrias and circadian-related metabolic disorders.

Main objectives and aims:

• Identify transient haem-binding sites in known haem chaperones such as GAPDH.

• Investigate the impact of haem trafficking on circadian rhythm regulation, including how haem binding modulates transcription factor activity.

• Elucidate the molecular mechanisms of haem exchange and transfer using structural biology techniques (e.g., X-ray crystallography, cryo-EM), biophysical methods (e.g., spectroscopy, binding assays), and computational modelling (e.g., molecular dynamics simulations).

• Explore implications for diseases associated with haem dysregulation, such as porphyrias or circadian disorders, to identify potential therapeutic targets.