Biology subjects

Hura, G. L.

Publications and source records attributed to Hura, G. L..

6 recordsLinked to original sources

bioRxiv2026

Programmable De Novo Design of Mesoporous Protein Crystal Frameworks

Three-dimensional protein crystals are ordered, porous macroscopic materials with potential applications in catalysis, biosensing, and biomedicine. However, most protein crystals are obtained by empirical screening, providing limited control over the lattice architecture, pore geometry or component composition that determine material function. Here, we present a modular strategy for the programmable design of highly porous, framework-like protein crystals using predefined protein-protein interactions. This strategy yielded over 30 distinct protein crystals, including single-component and multicomponent P213 and I213 lattices that grow to over 100 micrometers in size. Small-angle X-ray scattering and electron microscopy showed close agreement between experimental lattices and computational models. RFdiffusion-guided design generated isomorphous variants with matched lattice parameters, enabling coherent protein crystal alloys, epitaxial core-shell growth and reversible shell assembly. The designed crystals exhibit tunable mesoporous architectures, with limiting apertures of 2-18 nm, and support genetically encoded incorporation of fluorescent protein guests. These results establish a general route to programmable lattice engineering of protein crystals and position them as genetically encoded, compositionally tunable mesoporous materials.

synthetic biology↗

bioRxiv2026

Experimental Data Driven AI Framework for Flexible Protein Conformational Reconstruction

Deep learning has revolutionized structural biology by prediction with near experimental accuracy static protein folds from amino acid sequence alone. However, proteins function as dynamic ensembles of protein conformation states, and current sequence-only models often fail to capture the specific conformational states and heterogeneity dictated by cellular environments or ligand binding. While recent generative models can sample broad conformational landscapes, they remain unconstrained by physical reality, often hallucinating plausible but experimentally invalid states. Here, we present AlphaSAXS, an end-to-end framework that constrains artificial intelligence (AI) inference using Small Angle X-ray Scattering (SAXS) experimental solution scattering data. By integrating real-space pair distance distributions (P(r)) directly into the AlphaFold architecture, AlphaSAXS effectively steers the structural hypothesis toward the experimentally observed structures. We demonstrate that AlphaSAXS resolves documented failure modes of sequence-only models in Apo-Holo transitions, successfully distinguishing between states with identical sequences but distinct scattering profiles. Furthermore, we introduce a hybrid inference protocol that couples deep learning with biophysical hydration modeling, enabling the reconstruction of solution state protein ensembles compatible with experimental data. This work establishes a paradigm for experimentally guided AI, bridging the gap between probabilistic sampling and biophysical measurement.

biophysics↗

bioRxiv2023

The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the antiviral innate immune response

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non- structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5- triphosphorylated double-stranded RNA (5ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5ppp-dsRNA, and thus suppresses activation of RIG-Is ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

biochemistry↗

bioRxiv2023

Mapping the Morphology of DNA on Carbon Nanotube-Based Sensors in Solution using X-ray Scattering Interferometry

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes. However, it remains unclear how the ssDNA conformation on the SWCNT surface contributes to their ultimate functionality, as observations have been constrained to computational models or experiments under dehydrated states that differ substantially from the aqueous biological environments in which the nanosensors are applied. Herein, we demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two ssDNA oligomer lengths, conformational changes as a function of ionic strength, and the mechanism of dopamine sensing for a previously established ssDNA-SWCNT nanosensor, with corresponding ab initio modeling for visualization. We show that the shorter oligomer, (GT)6, adopts a highly ordered structure of stacked rings along the SWCNT axis, compared to the longer, less periodic (GT)15 wrapping. The presence of dopamine elicits a simultaneous axial elongation and radial constriction of the ssDNA closer to the SWCNT surface. Application of XSI to probe solution-phase morphologies of nanoparticle-based tools will yield insights into sensing mechanisms and inform future design strategies for polymer-functionalized SWCNT technologies.

biophysics↗

bioRxiv2022

Three-dimensional structure of the single domain cupredoxin AcoP

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

biophysics↗

bioRxiv2021

Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

biophysics↗