Biology subjects

Mendes, L. F. S.

Publications and source records attributed to Mendes, L. F. S..

10 recordsLinked to original sources

bioRxiv2026

Experimental Validation of Coiled-Coil Architecture and Folding Dynamics in the Golgin Bug1

Golgins are widely described as long coiled-coil proteins that contribute to the structural organisation and trafficking functions of the Golgi apparatus. Although experimental structures have been determined for a limited number of golgin regions, atomic-level information on their extended coiled-coil segments remains scarce, and the oligomeric state, topology, and register of most predicted regions remain unestablished. Here, we characterise a predicted coiled-coil region of the yeast golgin Bug1 (BUG1cc) using structural, biophysical, and computational approaches. X-ray crystallography revealed a parallel, in-register dimeric coiled-coil containing ten heptad repeats and a predominantly hydrophobic core, with specific polar interactions contributing to dimer stabilisation. In solution, BUG1cc was dimeric under SEC-MALS conditions and remained highly alpha-helical across the pH and ionic strength conditions examined. CD measurements revealed pronounced scan-rate-dependent hysteresis, while DSC independently confirmed an asymmetry between heating and cooling transitions. Increasing protein concentration shifted both apparent transition temperatures while preserving thermal hysteresis, supporting chain association and conformational rearrangements in structural recovery. Structure-based simulations indicated that interface contacts and intra-chain helicity are thermodynamically coupled and melt as a single cooperative unit, and that the monomer released on dissociation is compact and only partially helical, so that reassociation proceeds through a coupled folding-binding mechanism whose rate-limiting step is conformational rather than bimolecular. Together, these results establish the molecular architecture of a predicted coiled-coil region of Bug1 and reveal a complex folding landscape in which oligomerisation, secondary-structure recovery, and kinetic barriers are tightly coupled.

biophysics↗

bioRxiv2025

The role of N-terminal acetylation on biomolecular condensation

N-terminal acetylation (Nt-acetylation) is one of the most prevalent co-translational modifications in eukaryotes, affecting nearly 80% of the human proteome. Despite its ubiquity, the potential impact of this phenomenon on biomolecular condensation has been largely overlooked. Here, we uncover how this chemically subtle modification can exert broad and multifaceted control over phase behaviour, using Grh1, a Golgi-associated protein involved in stress-induced secretion in yeast, as a model system. We show that Nt-acetylation increases the saturation concentration for condensation, reduces droplet size and number, dampens pH sensitivity, weakens electrostatic contributions, and suppresses water dipolar relaxation within condensates, indicating reduced internal hydration and environmental responsiveness. These effects are accompanied by acetylation-dependent dimerisation and local structural changes, including a concentration-dependent gain in -helicity. Remarkably, co- condensation assays reveal that acetylated and non-acetylated forms of the same protein are only partially miscible, giving rise to core-shell architectures driven by differences in interfacial tension. Together, our findings highlight Nt-acetylation as a potent, generalizable regulator of condensate material properties, linking primary sequence chemistry to mesoscale organisation. Given its evolutionary conservation and prevalence across eukaryotic proteomes, Nt-acetylation may represent a widespread mechanism for modulating protein condensation in health and disease.

biophysics↗

bioRxiv2023

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion

Eukaryotic cells have developed sophisticated mechanisms for biomolecule transport, particularly under stress conditions. This study focuses on the unconventional protein secretion (UPS) pathways activated during starvation stress, facilitating the export of soluble leaderless proteins. Specifically, we examine the role of the GRASP family, which is crucial in these processes but has largely unexplored mechanisms, especially regarding the biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results reveal that under the circumstances mimicking starvation conditions in yeast, liquid-liquid phase separation (LLPS) is instrumental in the self-association of Grh1, the yeast homologue of GRASP. This association is likely a precursor to the biogenesis of the so-called Compartment for Unconventional Protein Secretion. We demonstrate that Grh1s self-association involves complex interactions and is regulated by electrostatic, hydrophobic, and hydrogen-bonding forces. Importantly, our study shows that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations, a significant finding given the minimal interaction between these proteins under normal conditions. Additionally, we address how droplet ageing might affect the ability of cells to return to normal post-stress states. Our findings can potentially enhance our understanding of intracellular protein dynamics and their adaptive responses to stress in eukaryotic cells.

biophysics↗

bioRxiv2023

Exploring the Theoretical Propensity for Liquid-Liquid Phase Separation in the Organization of Golgi Matrix Proteins

The Golgi apparatus is a critical organelle in protein sorting and lipid metabolism. Characterized by its stacked, flattened cisternal structure, the Golgi exhibits distinct polarity with its cis- and trans-faces orchestrating various protein maturation and transport processes. At the heart of its structural integrity and organization are the Golgi Matrix Proteins (GMPs), predominantly comprising Golgins and GRASPs. These proteins contribute to this organelles unique stacked and polarized structure and ensure the precise localization of Golgi-resident enzymes, which is crucial for accurate protein processing. Despite over a century of research since its discovery, the Golgi architectures intricate mechanisms still need to be fully understood. Here, we demonstrate that GMPs present a significant tendency to form biocondensates through Liquid-Liquid Phase Separation (LLPS) across different Eukaryotic lineages. Moreover, we validated experimentally that members of the GRASP family also exhibit a strong tendency for LLPS. Our findings offer a new perspective on the possible roles of protein disorder and LLPS of GMPs in the Golgi organization.

biochemistry↗

bioRxiv2023

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it an interesting player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. The possible application of these microreactors in bioremediation is discussed.

biophysics↗

bioRxiv2021

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the proteins secondary structure, according to our circular dichroism data, it had a significant impact on the proteins thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

biophysics↗

bioRxiv2021

Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics

The transmembrane emp24 domain-containing proteins (TMED), also called p24 proteins, are members of a family of sorting receptors present in all representatives of the domain Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMEDs structure, oligomerization propensity, and biophysics of their interactions with the transport cargo. Here, we describe the first high-resolution structure of the Golgi dynamics (GOLD) domain of a TMED1 representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane are also presented.

biophysics↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2019

Exploring Structural Aspects of the Human Golgi Matrix Protein GRASP55 in Solution

In mammalian cells, the Golgi apparatus is a central hub for intracellular trafficking, sorting and post-translational modifications of proteins and lipids. The Golgi reassembly and stacking proteins (GRASPs) are somehow involved in the Golgi stacking, which is significant for the proper function of the Golgi apparatus, and also in unconventional protein secretion. However, the structural details on how GRASPs accomplish those tasks are still elusive. In this context, we have explored the biochemical and biophysical properties of the human full-length GRASP55 in solution. Sequence-based analyses and circular dichroism spectroscopy suggest that GRASP55 presents multiple intrinsically disordered sites, although keeping considerable contents of secondary structure. Size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS) studies show that GRASP55 are monomers in solution. Urea denaturation of GRASP55 suggests that the transition to the unfolded state is a cooperative process. Differential scanning calorimetry (DSC) analysis displays two endothermic transitions for GRASP55, indicating the existence of an intermediate state prior to unfolding. Thioflavin T fluorescence shows that GRASP55 can form protein aggregates/fibrils at the intermediate state. Transmission electron microscopy and fluorescence lifetime imaging microscopy prove that GRASP55 forms large amorphous aggregates but not amyloid-like fibrils in the intermediate state. The significance of these results could be helpful in discussing the proper function of human GRASP55 in the Golgi organization as well as unconventional secretion of proteins.

biophysics↗

bioRxiv2018

The yeast GRASP Grh1 displays a high polypeptide backbone mobility along with an amyloidogenic behavior

GRASPs are proteins involved in cell processes that seem paradoxical, such as being responsible for shaping the Golgi cisternae and also involved in unconventional secretion mechanisms that bypass the Golgi, among other functions in the cell. Despite its involvement in several relevant cell processes, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that is also observed in the GRASP from the yeast S. cerevisae (Grh1), which strongly suggests it may be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and amyloid fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

biophysics↗