Biology subjects

Mravic, M.

Publications and source records attributed to Mravic, M..

6 recordsLinked to original sources

bioRxiv2026

De novo design of transmembrane accessory subunits for fold stabilization and expansion

Transmembrane (TM) proteins play essential roles in biology as transporters, ion channels, chaperones, enzymes, and mediators of signal transduction. However, membrane proteins often suffer from inefficient folding and intrinsic instability. Misfolding in cells can cause numerous loss-of-function pathologies. Likewise, denaturation upon purification in the laboratory is a critical barrier to structure determination and characterization of key biochemical mechanisms. Generalizable strategies to stabilize membrane proteins remain limited. Here, we developed an informatics-based de novo design strategy to create synthetic auxiliary subunits that interact with the TM helices of a model pentameric ion channel, thereby bolstering folding while maintaining channel function. Biochemical and structural characterization reveal the synthetic TM subunits can also be used to create larger multi-spanning designer proteins of custom topology. This proof-of-concept motivates the feasibility of computationally designed accessory TM helices as potential pharmacological chaperone "folding correctors" of membrane proteins in disease and as tools in structural biology.

biochemistry↗

bioRxiv2025

Design principles of the common Gly-X6-Gly membrane protein building block

Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X6-Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based designs X-ray structure hosts C-H{middle dot}{middle dot}{middle dot}O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SignificanceMembrane proteins comprised of -helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture ([~]10% of those in membrane folds). Small-X6-Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.

biophysics↗

bioRxiv2025

MemPPI platform for measuring and engineering membrane protein-protein interactions in mammalian cells via split nanoluciferase

Membrane protein-protein interactions (memPPIs) underlie critical biological processes including mediating signal transduction, transport, cell communication, and membrane organization. Despite their importance, systematic and quantitative measurement of protein transmembrane (TM) domains laterally interacting in mammalian cells remains challenging. Conventional fusion protein assays directed at memPPIs suffer from low dynamic range, due to the crowded membrane environment and interference from overexpression, trafficking, or self-complementation artifacts. Here, we assessed and further developed the split luciferase complementation NanoBit system to establish suitable expression constructs and working conditions for robust quantification of TM PPIs within human cells. We benchmarked the platform using a panel of natural and synthetic memPPI pairs with diverse architectures, insertion topologies, and trafficking patterns, finding assay settings that robustly distinguish true positive interactions from co-expressed non-interacting pairs. Adding a novel procedure measuring and normalizing protein expression levels in situ in parallel, we drastically improve assay dynamic range and consistency in discerning relative interaction propensities across broad expression levels - which also enhanced compatibility with protein engineering screens. To extend measurements to versatile protein contexts, we developed membrane expression constructs and validated tagging strategies to improve trafficking with defined TM orientations. By incorporating topological control and accounting for variations in expression levels, this memPPI platform becomes a viable approach for high-throughput interaction analyses and experimental screens for membrane protein design such as TM binders targeting human membrane proteins. Owing to its sensitivity, scalability, and adaptability, we expect this assay platform to have broad cross-disciplinary utility in protein science, design, and drug discovery.

bioengineering↗

bioRxiv2023

Ab initio prediction of specific phospholipid complexes and membrane association of HIV-1 MPER antibodies by multi-scale simulations

A potent class of HIV-1 broadly neutralizing antibodies (bnAbs) targets the envelope glycoproteins membrane proximal exposed region (MPER) through a proposed mechanism where hypervariable loops embed into lipid bilayers and engage headgroup moieties alongside the epitope. We address the feasibility and determinant molecular features of this mechanism using multi-scale modeling. All-atom simulations of 4E10, PGZL1, 10E8 and LN01 docked onto HIV-like membranes consistently form phospholipid complexes at key complementarity-determining region loop sites, solidifying that stable and specific lipid interactions anchor bnAbs to membrane surfaces. Ancillary protein-lipid contacts reveal surprising contributions from antibody framework regions. Coarse-grained simulations effectively capture antibodies embedding into membranes. Simulations estimating protein-membrane interaction strength for PGZL1 variants along an inferred maturation pathway show bilayer affinity is evolved and correlates with neutralization potency. The modeling demonstrated here uncovers insights into lipid participation in antibodies recognition of membrane proteins and highlights antibody features to prioritize in vaccine design.

biophysics↗

bioRxiv2023

Designed Transmembrane Proteins Inhibit the Erythropoietin Receptor in a Custom Binding Topology

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom designed topologies.

bioengineering↗

bioRxiv2022

Transient Water Wires Mediate Selective Proton Conduction in Designed Channel Proteins

Summary paragraphSelective proton transport through proteins is essential for forming and utilizing proton gradients in cells. Protons are conducted along hydrogen-bonded "wires" of water molecules and polar sidechains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways inferred from static protein structures. We hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess proton itself in the water wire. To test this hypothesis, we used molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 106-fold more selective for H+ over Na+. These studies inform mechanisms of biological proton conduction and principles for engineering proton-conductive materials.

biophysics↗