Biology subjects

Souza, P. C. T. d.

Publications and source records attributed to Souza, P. C. T. d..

5 recordsLinked to original sources

bioRxiv2026

3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

biophysics↗

bioRxiv2024

AlphaFold-Multimer struggles in predicting PROTAC-mediated protein-protein interfaces

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules composed by ligands binding to a target protein and a E3-ligase complex, connected by a linker, that induce proximity-based target protein degradation. PROTACs are promising alternatives to conventional drugs against cancer. Predicting PROTAC-mediated complexes is often the first step for in silico PROTAC design pipelines. AlphaFold2 (AF2) revolutionized structural biology, enabling the prediction of multimeric protein structures. However, we previously noted that AF2 fails to predict PROTAC-mediated complexes. Here, we investigate the potential causes of this limitation. We consider a set of 326 protein heterodimers orthogonal to the AF2 training set, and evaluate AF2 models focusing on the interface size and presence of interface ligand. Our results show that AF2-multimer predictions are sensitive to the size of the interface to predict even in the absence of ligands, with the majority of models being incorrect for the smallest interfaces. We also benchmark both AF2 and AF3 on a set of 28 PROTAC-mediated dimers and show that AF3 does not significantly improve upon the accuracy of AF2. The low accuracy of AF2 on complexes with small interfaces has strong implications for computational pipelines for PROTAC design, as these stabilize typically small interfaces, and more generally on any prediction task that involves small interfaces.

bioinformatics↗

bioRxiv2024

Cholesterol modifies Nav1.7 - an in-silico and in-vitro analysis

Cholesterol is a major component of plasma membranes and unsurprisingly plays a significant role in actively regulating the functioning of several membrane proteins in humans. Notably, recent studies have shown that cholesterol depletion can also impact transmission of potentially painful signals in the context of peripheral inflammation, via hyperexcitability of the voltage-gated sodium channel (Nav) subtype 1.9, but the structural mechanisms underlying this regulation remain to be elucidated. In this study, we focus on the role of cholesterol depletion on Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations shed light on the dynamic changes of the geometry of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity at key structural motifs linked to activation and fast-inactivation is observed, as well as changes in the geometry of drug-binding regions in the channel. Loss of rigidity in cholesterol depleted conditions should allow the channel to transition between different gating states more easily. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions made in silico at the functional level. Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results hint to a key role of the membrane environment in affecting drug effects and in pathophysiological dysregulation, sharpening our approaches for analgesics design. Supplementary datahttps://doi.org/10.5281/zenodo.10829175

physiology↗

bioRxiv2023

A millisecond coarse-grained simulation approach to decipher allosteric cannabinoid binding at the glycine receptor α1

Glycine receptors (GlyR) are regulated by small-molecule binding at several allosteric sites. Cannabinoids like tetrahydrocannabinol (THC) and N-arachidonyl-ethanol-amide (AEA) potentiate GlyR but their mechanism of action is not fully established. By combining millisecond coarse-grained MD simulations powered by Martini 3 with backmapping to all-atom representations, we characterize the cannabinoid-binding sites at zebrafish GlyR-1 with atomic resolution. Based on hundreds of thousand ligand-binding events, we find that cannabinoids bind to the transmembrane domain of the receptor at both intrasubunit and intersubunit sites. For THC, the intrasubunit binding mode predicted in simulation is in excellent agreement with recent cryo-EM structures, while intersubunit binding recapitulates in full previous mutagenesis experiments. Intriguingly, AEA is predicted to bind at the same intersubunit site despite the strikingly different chemistry. Statistical analyses of the receptor-ligand interactions highlight potentially relevant residues for GlyR potentiation, offering experimentally testable predictions. The predictions for AEA are validated by electrophysiology recordings of rationally designed mutants. The results highlight the existence of multiple cannabinoid-binding sites for the allosteric regulation of GlyR and put forward an effective strategy for the identification and structural characterization of allosteric sites in transmembrane proteins.

biophysics↗

bioRxiv2020

Bottom-up fabrication of a multi-component nanopore sensor that unfolds, processes and recognizes single proteins

Transmembrane channels and pores have many biotechnological applications, notably in the single-molecule sequencing of DNA. Small synthetic nanopores have been designed using amphipathic peptides, or by assembling computationally designed transmembrane helices. The fabrication of more complex transmembrane devices has yet to be reported. In this work, we fabricated in two steps a multi-protein transmembrane device that addresses some of the main challenges in nanopore protein sequencing. In the first step, artificial nanopores are created from soluble proteins with toroid shapes. This design principle will allow fabricating a variety of nanopores for single-molecule analysis. In the second step one -subuinit of the 20S proteasome from Thermoplasma acidophilum is genetically integrated into the artificial nanopore, and a 28-component nanopore-proteasome is co-assembled in E. coli cells. This multi-component molecular machine opens the door to two new approaches in protein sequencing, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then identified by the nanopore sensor either as intact or fragmented polypeptides. The ability to integrate molecular devices directly onto a nanopore sensors allows creating next-generation protein sequencing devices, and will shed new lights on the fundamental processes of biological nanomachines.

bioengineering↗