Biology subjects

Hopkins, J. B.

Publications and source records attributed to Hopkins, J. B..

11 recordsLinked to original sources

bioRxiv2026

Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

bioengineering↗

bioRxiv2024

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

biophysics↗

bioRxiv2024

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 [A]. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

biophysics↗

bioRxiv2024

A General Mechanism for the General Stress Response in Bacteria

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

microbiology↗

bioRxiv2023

Design of intrinsically disordered protein variants with diverse structural properties

Intrinsically disordered proteins (IDPs) perform a wide range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Designing IDPs with specific structural or functional properties has, however, been diffcult, in part because determining accurate conformational ensembles of IDPs generally requires a combination of computational modelling and experiments. Motivated by recent advancements in effcient physics-based models for simulations of IDPs, we have developed a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs with different levels of compaction and that vary more than 100 fold in their propensity to undergo phase separation, even while keeping a fixed amino acid composition. We experimentally tested designs of variants of the low-complexity domain of hnRNPA1 and find high accuracy in our computational predictions, both in terms of single-chain compaction and propensity to undergo phase separation. We analyze the sequence features that determine changes in compaction and propensity to phase separate and find an overall good agreement with previous findings for naturally occurring sequences. Our general, physics-based method enables the design of disordered sequences with specified conformational properties. Our algorithm thus expands the toolbox for protein design to include also the most flexible proteins and will enable the design of proteins whose functions exploit the many properties afforded by protein disorder.

biophysics↗

bioRxiv2023

BioXTAS RAW 2: new developments for a free open-source program for small angle scattering data reduction and analysis

BioXTAS RAW is a free, open-source program for reduction, analysis and modelling of biological small angle scattering data. Here, the new developments in RAW version 2 are described. These include: improved data reduction using pyFAI; updated automated Guinier fitting and Dmax finding algorithms; automated series (e.g. SEC-SAXS) buffer and sample region finding algorithms; linear and integral baseline correction for series; deconvolution of series data using REGALS; creation of electron density reconstructions via DENSS; a comparison window showing residuals, ratios, and statistical comparisons between profiles; and generation of PDF reports with summary plots and tables for all analysis. In addition, there is now a RAW API, which can be used without the GUI, providing full access to all of the functionality found in the GUI. In addition to these new capabilities, RAW has undergone significant technical updates, such as adding Python 3 compatibility, and has entirely new documentation available both online and in the program.

biophysics↗

bioRxiv2023

The PALB2 DNA binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

biophysics↗

bioRxiv2023

Structural context of homomeric interactions in the Ig domain of the MPZ (P0) myelin adhesion protein and relation to Charcot-Marie-Tooth disease phenotype variants

Mutations in Myelin Protein Zero (MPZ) account for 5% of Charcot-Marie-Tooth cases and can cause demyelinating or axonal phenotypes, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds the apposing membranes of the myelin sheath together, with the adhesion role fulfilled by the extracellular lmmunoglobulin-like domain (lgMPZ), which can oligomerize. Current knowledge for how the lgMPZ might form oligomeric assemblies involving 3 weakly-interacting interfaces has been extrapolated from a protein crystal structure in which individual rat lgMPZ subunits are packed together under artificial conditions. These interfaces include one that organizes the lgMPZ into tetramers, a dimer interface that could link tetramers together, and a third hydrophobic interface that could mediate binding to lipid bilayers or the same hydrophobic surface on another lgMPZ domain. There are at present no data confirming whether the proposed lgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed NMR and SAXS analysis of wild-type lgMPZ as well as mutant forms with amino-acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates lgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated CMT phenotypes to subregions within lgMPZ tetramers. Axonal late-onset disease phenotypes (CMT2l/J) map to surface residues of lgMPZ proximal to the transmembrane domain. Early-onset demyelinating disease phenotypes (CMT1B/Dejerine-Sottas syndrome) map to two groups: one is described by variants that disrupt the stability of the lg-fold itself and are largely located within the core of the lg domain; whereas another describes a surface on the distal outer surface of lgMPZ tetramers. Computational docking studies predict that this latter disease-relevant subregion may mediate dimerization of lgMPZ tetramers.

neuroscience↗

bioRxiv2022

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic with breakdown by gut bacteria requiring the action of specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses granular starch-binding protein Sas6 (Starch Adherence System member 6) that consists of two starch-specific carbohydrate binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here we present the crystal structures of Sas6 and RbCBM74 with a double helical dimer of maltodecaose bound along an extended surface groove. Binding data combined with native mass spectrometry suggest that RbCBM26 binds short maltooligosaccharides while RbCBM74 can bind single and double helical -glucans. Our results support a model by which RbCBM74 and RbCBM26 bind neighboring -glucan chains at the granule surface. CBM74s are conserved among starch granule-degrading bacteria and our work provides molecular insight into how this structure is accommodated by select gut species.

molecular biology↗

bioRxiv2022

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its [~]30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate. TeaserWe elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.

biochemistry↗

bioRxiv2021

Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Here, we found that, although some polyubiquitin chain types (K11-Ub4 and K48-Ub4) did generally inhibit UBQLN2 LLPS, others (K63-Ub4, M1-Ub4 and a designed tetrameric ubiquitin construct) significantly enhanced LLPS. Using nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in chain conformations, but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin binding surface significantly promoted UBQLN2 LLPS by enabling a switch between homotypically to partially heterotypically-driven phase separation. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic phase separation with ubiquitin-binding shuttles and adaptors. HighlightsO_LIUbiquitin or short polyubiquitin chains bind to phase separation-driving stickers on UBQLN2 and inhibit its phase separation whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation. C_LIO_LIPhase separation of UBQLN2 is promoted over a wide range of Ub:UBQLN2 ratios in the presence of extended M1- and K63-linked Ub4 chains, but not compact K11- and K48-linked Ub4 chains. C_LIO_LIChain conformation and accessibility of the Ub interacting surface is a driving factor of UBQLN2/polyUb co-phase separation. C_LIO_LIUBQLN2 condensates assemble during in vitro enzymatic assembly of K63-linked polyUb chains as free ubiquitin is reduced. C_LI

biophysics↗