Biology subjects

Porcar, L.

Publications and source records attributed to Porcar, L..

8 recordsLinked to original sources

bioRxiv2026

Calibrated ADAMTS13 inhibition prevents cardiovascular shear coagulopathy

Mechanical circulatory support essential for managing severe heart failure frequently triggers bleeding complications, driven by shear stress-induced over-proteolysis of von Willebrand factor (VWF) by ADAMTS13. Inhibiting ADAMTS13 presents a rationale to treat this condition, known as acquired von Willebrand syndrome (AVWS). However, conventional therapeutic strategies remain limited due to the risk of triggering thrombotic thrombocytopenic purpura. Here we show that HA10, a humanized anti-ADAMTS13 antibody, preserves a residual level of ADAMTS13 activity above the thrombosis-associated threshold. Multimodal structural and biophysical analyses--including NMR, SAXS, and SANS--revealed that HA10 bound to the disintegrin-like domain of ADAMTS13, dynamically competing with VWF while leaving 10- 20% residual enzymatic activity. The therapeutic efficacy and safety of HA10 were verified in non-human primate models of AVWS. Our findings establish a novel paradigm of enzymatic calibration rather than complete blockade, offering a mechanistically targeted and safe therapeutic approach for cardiovascular bleeding.

biochemistry↗

bioRxiv2026

Integrating Segmental Deuteration iCM-SANS with SAXS and MD for Dynamical Analysis of Multi-domain Proteins

Multi-domain proteins (MDPs) adopt diverse conformations arising from cooperative inter-domain motions, and such dynamics are intimately coupled to their biological functions. Quantitative characterization of these motions is crucial for elucidating their functional mechanisms. Although small-angle X-ray scattering (SAXS) provides information on overall domain arrangement, the limited experimental constraints hinder reliable discrimination of conformational ensembles derived from molecular dynamics (MD) simulations. To address this limitation, complementary experimental constraints that enable to observe domain-selective structural information are required. Inverse contrast-matching small-angle neutron scattering (iCM-SANS), combined with segmental deuteration, enables selective visualization of individual domains and thus provides such complementary information. However, practical strategies for preparing segmentally deuterated MDPs with arbitrary domain labelling have yet to be established. Here, we develop an experimental protocol that integrates controlled protein deuteration with high-efficiency multi-step protein ligation to generate a segmentally deuterated MDP in high yield. The combined use of SAXS and iCM-SANS yields complementary structural constraints that enhance discrimination of MD-derived conformational ensembles. This protocol expands the applicability of segment-selective visualization and also provides an opportunity for high-precision analysis of dynamics in complex MDPs. SynopsisSegmental deuteration enabled by high-efficiency multi-step protein ligation, combined with inverse contrast-matching SANS and SAXS, provides structural constraints that improve discrimination of molecular dynamics ensembles of multi-domain proteins. IMPORTANTthis document contains embedded data - to preserve data integrity, please ensure where possible that the IUCr Word tools (available from http://journals.iucr.org/services/docxtemplate/) are installed when editing this document.

biophysics↗

bioRxiv2024

Hierarchical structural organization in bioinspired peptide coacervate microdroplets

This study explores the dynamic and hierarchical structural organization of peptide coacervate microdroplets at the meso-to atomic-scale resolution using a combination of Transferred Nuclear Overhauser Effect Spectroscopy (TrNOESY), Small Angle Neutron Scattering (SANS), and confocal microscopy. Dynamic interactions driving the self-association of peptide clusters are revealed, highlighting the critical roles of interacting residues. These phase-separating model peptides form small oligomers at low pH, which aggregate into larger clusters at neutral pH. These clusters organize into a porous network within the droplets, facilitating size-selective cargo sequestration. The findings underscore the significance of the dynamic spatio-temporal properties of peptide-based coacervates, contributing to our understanding of phase separation at the atomic and molecular levels. Critically, this approach enables the investigation of coacervate structures in their native state, offering insights into the physical and dynamic interactions governing droplet formation and cargo encapsulation. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/602323v4_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@15aadb2org.highwire.dtl.DTLVardef@41be55org.highwire.dtl.DTLVardef@36f0d8org.highwire.dtl.DTLVardef@fe1af8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Distributing Aminophospholipids Asymmetrically Across Leaflets Causes Anomalous Membrane Stiffening

We studied the mechanical leaflet coupling of prototypic mammalian plasma membranes using neutron spin-echo spectroscopy. In particular, we examined a series of asymmetric phospholipid vesicles with phosphatidylcholine and sphingomyelin enriched in the outer leaflet and inner leaflets composed of phosphatidylethanolamine / phosphatidylserine mixtures. The bending rigidities of most asymmetric membranes were anomalously high, exceeding even those of symmetric membranes formed from their cognate leaflets. Only asymmetric vesicles with outer leaflets enriched in sphingolipid displayed bending rigidities in conformity with these symmetric controls. We performed complementary small-angle neutron and X-ray experiments on the same vesicles to examine possible links to structural coupling mechanisms, which would show up in corresponding changes in membrane thickness. Additionally, we estimated differential stress between leaflets originating either from a mismatch of their lateral areas or spontaneous curvatures. However, no correlation with asymmetry-induced membrane stiffening was observed. To reconcile our findings, we speculate that an asymmetric distribution charged or H-bond forming lipids may induce an intraleaflet coupling which increases the weight of hard undulatory modes of membrane fluctuations and hence the overall membrane stiffness. O_TEXTBOXSIGNIFICANCE Biological membranes not only act as the outer barriers of cells and organelles, but host a delicate machinery of proteins, which is sensitive not only to the surrounding lipid composition, but also to the mechanical properties of the bilayer. Little is known about the effect of transbilayer lipid asymmetry, a hallmark of all plasma membranes, on bilayer structure and dynamics. The results of this study indicate that the composition of the plasma membrane leads to unique mechanical coupling mechanisms, suggesting that the regulation of lipid asymmetry through flipases/flopases and scramblases could be an important means to tune the mechanical stiffness of the membrane. C_TEXTBOX

biophysics↗

bioRxiv2022

Human myelin proteolipid protein structure and lipid bilayer stacking

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, -helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

biochemistry↗

bioRxiv2021

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

biophysics↗

bioRxiv2019

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In the present study, the solution structure of the OLDN was investigated through integration of small-angle X-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis, and searched for the conformations that well reproduced the SAXS and SANS scattering curves. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.\n\nStatement of SignificanceOverlapping dinucleosomes (OLDNs) are intermediate structures formed through nucleosome collision promoted by nucleosome remodeling factor(s). To study the solution structure of OLDNs, a structural library containing a wide variety of conformations was prepared though simulations, and the structures that well reproduced the small angle X-ray and neutron scattering data were selected from the library. Simultaneous evaluation of the conformational variation in the global OLDN structures and in the histone tails is difficult using conventional MD simulations. We overcame this problem by combining multiple simulation techniques, and showed the importance of the histone tails for stabilizing the structures of OLDNs in solution.

biophysics↗

bioRxiv2018

Direct Evidence of Effect of Glycerol on Hydration and Helix-to-Sheet Transition of Myoglobin

By using wide-angle X-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation, we have clarified the effect of glycerol on both the thermal structure transition and the hydration-shell of myoglobin. At the glycerol concentration, [≤] [~]40 % v/v, the decreasing tendency in the maximum dimension and the radius of gyration was observed by X-ray scattering. The neutron scattering result using the inverse contrast variation method directly shows the preservation of the hydration-shell density at the concentration [≤] [~]40 % v/v. This phenomenon is reasonably explained by the preferential exclusion of glycerol from the protein surface to preserve the hydration shell, as suggested by the previous studies. While, at the concentration, [≥] 50 % v/v, the opposite tendency was observed. It suggests the preferential solvation (partial preferential penetration or replacement of glycerol into or with hydration-shell water surrounding the protein surface) occurs at the higher concentration. The observed WAXS scattering data covers the distinct hierarchical structural levels of myoglobin structure ranging from the tertiary structure to the secondary one. Therefore, we have clarified the effect of glycerol on the thermal structural stability myoglobin at different hierarchical structural levels separately. Against the temperature rise, the structural transition temperatures for all hierarchical structural levels were elevated. Especially, the tertiary structure of myoglobin was more stabilized compared with the internal-structure and the helix-to-cross transition. It suggests that the protective action of glycerol on protein structures essentially results from the preservation of the preferential hydration-shell of proteins.

biophysics↗